3D microphysiologic system

ABSTRACT

The present invention relates generally to a three-dimensional cell and tissue culture system for the female reproductive tract. In particular provided herein the system includes individual female reproductive cultures in a dynamic microfluidic setting or integrated using a microfluidic microphysiologic system. In some embodiments, the present invention provides ex-vivo female reproductive tract integration in a three dimensional (3D) microphysiologic system.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/932,592, filed Jan. 28, 2014, which isincorporated by reference in its entirety.

STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under ES022920 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD

The present invention relates generally to a three-dimensional cell andtissue culture systems for the female reproductive tract. In particularprovided herein are the system includes individual female reproductivecultures integrated using a microfluidic microphysiologic system.

BACKGROUND

The main organs of reproductive tract include the ovary, fallopiantubes, uterus, cervix, and vagina. These organs function in relation toone another to provide hormonal support or the anatomical structurethrough which gametes travel for the developing embryo to implant anddevelop. A robust three dimensional reproductive tract that is aphysiologic mimic of the in vivo biology is what is needed.

SUMMARY

The present invention relates generally to a three-dimensional cell andtissue culture systems for the female reproductive tract. In particular,provided herein are systems comprising individual female reproductivecultures integrated using a microfluidic microphysiologic system. Insome embodiments, the present invention provides ex-vivo femalereproductive tract integration in a three dimensional (3D)microphysiologic system.

In some embodiments, provided herein are microphysiologic systems (e.g.,systems that simulate one or more aspects of the human reproductivetract) comprising: (a) a first 3D cell culture subsystem comprising atleast a first cell type in 3D culture and a culture media for said firstcell type; and (b)

a second 3D cell culture subsystem comprising at least a second celltype in 3D culture and a culture media for said second cell type;wherein the first 3D cell culture subsystem and the second 3D cellculture subsystem are in unidirectional fluid communication such thatfactors secreted from said first cell type flow downstream to the second3D cell culture subsystem. In some embodiments, microphysiologic systemsfurther comprise one or more additional 3D cell culture subsystems, eachcomprising at least one additional and distinct cell type in 3D cultureand a culture media for said additional cell type. In some embodiments,microphysiologic systems further comprise a third 3D cell culturesubsystem comprising at least a third cell type in 3D culture and aculture media for said third cell type; wherein the second 3D cellculture subsystem and the third 3D cell culture subsystem are inunidirectional fluid communication such that factors secreted from saidsecond cell type and/or said first cell type flow downstream to thethird 3D cell culture subsystem. microphysiologic systems furthercomprise a fourth 3D cell culture subsystem comprising at least a fourthcell type in 3D culture and a culture media for said fourth cell type;wherein the third 3D cell culture subsystem and the fourth 3D cellculture subsystem are in unidirectional fluid communication such thatfactors secreted from said third cell type, said second cell type,and/or said first cell type flow downstream to the fourth 3D cellculture subsystem. In some embodiments, microphysiologic systems furthercomprise a fifth 3D cell culture subsystem comprising at least a fifthcell type in 3D culture and a culture media for said fifth cell type;wherein the fourth 3D cell culture subsystem and the fifth 3D cellculture subsystem are in unidirectional fluid communication such thatfactors secreted from said fourth cell type, said third cell type, saidsecond cell type, and/or said first cell type flow downstream to thefourth 3D cell culture subsystem. In some embodiments, the first 3D cellculture subsystem comprises an ovarian follicle 3D cell culturesubsystem, wherein the second 3D cell culture subsystem comprises afallopian tube 3D cell culture subsystem, wherein the third 3D cellculture subsystem comprises a uterine 3D cell culture subsystem, whereinthe fourth 3D cell culture subsystem comprises an endocervical 3D cellculture subsystem, and wherein the fifth 3D cell culture subsystemcomprises an ectocervical 3D cell culture subsystem. In someembodiments, the ovarian follicle 3D cell culture subsystem comprisesone or more of granulosa cells, theca cells, and oocytes. In someembodiments, the fallopian tube 3D cell culture subsystem comprises oneor more of secretory epithelial cells, ciliated epithelial cells, andfallopian stromal cells. In some embodiments, the uterine 3D cellculture subsystem comprises one or more of endometrial epithelial cells,endometrial stromal cells, myometrial smooth muscle cells, andmyometrial stromal cells. In some embodiments, the endocervical 3D cellculture subsystem comprises one or both of endocervical epithelial cellsand endocervical stromal cells. In some embodiments, the ectocervical 3Dcell culture subsystem comprises one or both of ectocervical epithelialcells, J2-3T3 fibroblasts, and ectocervical stromal cells. In someembodiments, the microphysiologic system comprises human or murinecells. In some embodiments, the ovarian follicle 3D cell culturesubsystem comprises one or more ovarian follicles in 3D culture. In someembodiments, the ovarian follicles are polymer-encapsulated orhydrogel-encapsulated. In some embodiments, the polymer or hydrogelcomprises alginate. In some embodiments, the ovarian follicles (or othercultured cells or tissues) are encapsulated or supported by a matrix orsupport material. Suitable matrices and/or support materials mayinclude, but are not limited to alginate, polyethylene glycol,poly(octanediol citrate), decellularized matrix (e.g., general orspecific), fibroblast-derived native matrix, collagen, matrigel,hyaluronan, laminin, entactin, tenascin, fibronectin, poly-1-lysine,fibrin, polystyrene scaffold (e.g., ALVETEX), etc. In some embodiments,the ovarian follicles remain viable for at least one simulated menstrualcycle. In some embodiments, the ovarian follicles respond to FSH and hCGstimulation by producing estrogen and progesterone in a pattern thatmimics the human menstrual cycle. In some embodiments, estrogen andprogesterone pass by unidirectional fluid communication to the fallopiantube 3D cell culture subsystem (e.g., from the ovarian follicle 3D cellculture subsystem). In some embodiments, the cells of the fallopian tube3D tissue culture subsystem respond to said estrogen and/or progesteronewith one or more of cilliary beating, OVGP1 expression, and/or IGF1secretion. In some embodiments, the fallopian tube 3D tissue culturesubsystem comprises fallopian epithelium tissue pieces grown onTRANSWELL inserts. In some embodiments, the fallopian tube systemremains viable and maintains both secretory and ciliated epithelium cellphenotypes for at least one simulated menstrual cycle. In someembodiments, systems (and/or subsystems) comprise a microfluidic systemthat provides said unidirectional fluid communication.

In other embodiments, one or more pairs of subsystems are inbidirectional fluid communication. In some embodiments, additionalfluids (e.g., media, comprising test compounds, etc.) are mixed with thefluid within the system at one or more positions in the system.

In some embodiments, the female-reproductive-tract-simulating-systemsfurther comprise or are integrated with (e.g., in fluid communicationwith) cultures (e.g., 3D cultures) of related or non-reproductive cellsor tissues. Suitable cells or tissues may include, but are not limitedto those from liver, lung, breast, skin, eye, adipose, bone, bloodvessel, myometrium, endometrium, placenta, etc. In some embodiments,cultures comprising such cells/tissues are in fluid communication (e.g.,bidirectional, unidirectional, continuous, with mixing, without mixing,etc.) with one or more of the other subsystems described herein. In someembodiments, methods of assessing the impact of these cells/tissues onreproductive cells/tissues and/or the menstrual cycle are provided. Insome embodiments, methods are provided of assessing the impact ofreproductive cells/tissues and/or the menstrual cycle on thesecells/tissues.

In some embodiments, systems described herein further comprise cultures(e.g., 3D culture) comprising one or more types of diseased cells ortissues. Diseased cells or tissues may be included in one of thesubsystems described herein (e.g., ovarian, fallopian, uterine,endocervical, ectocervical, etc.) or may be provided as an additionsubsystem integrated with (e.g., in fluid communication with) one of thesubsystems described herein. Exemplary disease cells/tissues includethose derived from uterine fibroid tissue, cancer tissue (e.g., ovarian,uterine, cervical, breast, liver, etc.), endometiotic tissue, pelvicinflammatory disease tissue, polycystic ovarian tissue, virally infectedtissue (e.g., ectocervix barrier function-disease transmission), variousmicrobiomes, etc.

In some embodiments, cultured cells/tissues are derived from inducedpluripotent stem cells (iPSCs). In some embodiments, two or more of thesubsystems comprise tissue/cells derived from the iPSCs from a subject.In some embodiments, such a system is used to assess the effects ofvarious agents (e.g., drugs, chemotherapeutics, allergens, environmentaltoxins, etc.) on that subject. In some embodiments, personalizedmedicine methods are provided by screening therapies via the systems andmethods described herein before administering to a subject.

In some embodiments, the systems and methods described herein are usedto simulate the female reproductive tract during particular states, forexample: pregnancy (e.g., comprising cultures of myometrium,endometrium, placenta, etc.; under appropriate hormone exposure; etc.),cancer or other diseases, during exposure to various agents (e.g.,hormones, chemotherapeutics or other drugs, environmental toxins),normal menstrual cycle, irregular cycles, etc.

In some embodiments, effects of various stimuli on thehuman-female-simulating systems described herein are compared to one ormore of: a human subject (e.g., a human female, a human male), anothersimulation of human biology, a simulation of the male reproductivesystem.

The present invention relates generally to a human female reproductivetract using individual three-dimensional tissue culture systems designedfor integration using a microfluidic microphysiologic system (FemKube).In certain embodiments, the system includes individual three dimensionalcultures of ovarian follicles (OvaryKube), fallopian tube (TubeKube),uterus (UteroKube), and cervix (CerviKube). In other embodiments, threedimensional cultures for the ectocervix and endocervix are providedrather than a single cervix culture. In some embodiments, the cultersare in fluid communication, such that hormones and other molecularcomponents of the system can pass (e.g., bidirectionally,unidirectionally, continually, with mixing, without mixing, etc.)between cultures. Importantly, each tissue remains viable for at least28 days and responds to hormonal fluctuations that mimic the humanmenstrual cycle. Prior 3D culture systems for female reproductive tracteither did not exist, used non-human cells, or were not relevant tolong-term (e.g., full menstrual cycle) physiologic processes. Inparticular embodiments, systems described herein use hormones secretedby the 3D cultured ovarian follicles to stimulate the other reproductivetissues rather than adding, for example, exogenous hormones to the cellculture media.

The present invention describes a system comprised of multiple (e.g. 2,3, 4, 5, 6, 7, 8, or more) individual female reproductivethree-dimensional cultures integrating the tissues using a microfluidicmicrophysiologic system (or other integrative system that allows fluidand/or analyte exchange between cultures). In certain embodiments, theindividual cultures are ovarian follicles, fallopian tube, uterus, andcervix. In other embodiments, the individual cultures are ovarianfollicles, fallopian tube, uterus, endocervix, and ectocervix.

In some embodiments, the ovarian culture system uses, for example, humanor murine follicles (e.g., encapsulated in alginate or other polymer).The follicles may be encapsulated in a substrate, matrix, polymer, etc.or may be unencapsulated. The (encapsulated) follicles remain viablelong term (e.g., one menstrual cycle (e.g., 28 days), more than onemenstrual cycle (e.g., 30-50 days), two menstrual cycles (e.g., 56days), more than two menstrual cycles (e.g., >56 days), etc.). Thefollicles of the ovarian culture system respond to signals found in invivo ovarian systems (e.g., follicle stimulating hormone (FSH) and humanchorionic growth hormone (hCG, etc.). In some embodiments, the folliclesundergo in-vitro maturation. In some embodiments, the follicles produceestrogen and progesterone in a pattern that mimics the human menstrualcycle. In certain embodiments, the fallopian tube system uses humanfallopian epithelium tissue pieces grown on TRANSWELL inserts. Tissueremains viable for 28 days and maintains both secretory and ciliatedepithelium cell phenotypes. Furthermore, the fallopian epitheliumfunctionally responds to estrogen and progesterone using secretedfactors and cilia beating as markers. The uterine system is comprised ofhuman endometrial epithelial, endometrial stromal, and myometrial cells.In certain embodiments, the endometrial epithelial and stromal cells areisolated separately and combined for culture on TRANSWELL inserts, whilemyomertial smooth muscle cells are cultured on a separate TRANSWELLinsert. In some embodiments, the endometrial and myometrial inserts arecultured in the same tissue culture well in a common media. In someembodiments, uterine cultures are viable for at least 28 days. In someembodiments, cervical cultures are comprised of primary humanendocervical epithelial and stromal cells (e.g., grown on the sameTRANSWELL insert). In some embodiments, endocervix remains viable for atleast 28 days and responds to estrogen and progesterone mimicking thehuman menstrual cycle.

In some embodiments of the invention, the organ culture system is usedto screen therapeutic compositions, to assess efficacy of apharmaceutical composition, to assess pharmaceutical toxicity, to assesstoxicity of non-pharmaceutical compositions, to assess toxicity ofenvironmental contaminants, to assess contraceptive compositions andmethods, for reproductive biology studies, to study normal femalereproductive tract biology, to study diseased female reproductive tractbiology, to study the female menstrual cycle, etc.

In some embodiments, provided herein is the use of systems andsubsystems described herein for testing the efficacy and/or toxicity ofthe pharmaceutical composition.

In some embodiments, provided herein is the use of systems andsubsystems described herein for testing the effects of various agents onone or more tissues (e.g., the entire reproductive tract). Suitableagents may comprise environmental toxins, pharmaceuticals (e.g.,chemotherapeutics, birth control, etc.), hormones, etc. Exemplary agentsinclude, but are not limited to: estradiol, ulipristal acetate, RU486,insulin, diethylstibestrol (DES), corexit 9500, medroxyprogesteroneacetate (MPA), follicle-stimulating hormone (FSH), human chorionicgonadotropin (hCG), progesterone, bisphenol A (BPA), testosterone,cisplatin, nalbuphine, raclopride, arsenic trioxide, nonoxynol-9,vitamin A, vitamin D, nicotine, glucose (e.g., to mimic a diabeticstate), caffeine, cortisol (stress), soy (e.g., including genistein),ethanol, etc.

Although some embodiments are described herein as related to systemssimulating the biological functions of the “human” female reproductivetract, the present invention is not so limited. In some embodiments,provided herein are microphysiologic systems that simulate one or moreaspects of the reproductive tract of a non-human animal (e.g., rodent,non-human primate, feline, canine, bovine, equine, porcine, etc.).

In some embodiments, systems are provided comprising a single 3D culture(e.g., described elsewhere herein as a subsystem (e.g., ovarianfollicles, fallopian tubes, uterus, endocervix, ectocervix, etc.) andmicrofluidics to provide a flow of media, hormones, factors, testagents, etc. In some embodiments, the microfluidics provides a flowmedia that simulates the upstream tissues. In some embodiments, flowfrom the culture is analyzed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Steroid and peptide hormone profiles of in vitro follicle growth(IVFG) cultured secondary human follicles. Steroid and peptide hormoneswere quantified in the medium throughout the culture period. Secondaryhuman follicles were isolated and cultured in alginate from 40 to 65days. The culture time on X-axis had been aligned to the time of the hCGaddition (day 0) and cultured for an additional 14-15 days. Theconcentration of (A) estradiol, (B) inhibin A, (C) AMH, (D)progesterone, (E) inhibin B, and (F) activin A are reported throughoutculture relative to hCG administration. Gray shading represents the invitro luteal phase, or time post-hCG. Follicles were derived from threedonors and individually cultured.

FIG. 2. Alginate culture supports human follicle growth and structuralluteinization. (A) Representative image of in vitro human folliclegrowth. A single secondary human follicle (147 μm) was isolated from theovarian cortex and grown in 0.3% alginate. On day 32, the folliclemeasured approximately 600 μm in diameter and hCG was administered. (B)Representative images of H&E stained secondary human follicles (i)pre-hCG and (ii) 15 days post-hCG (Culture days 32 and 43,respectively). Follicles showed morphological signs of luteinization,marked by a significant increase in cytoplasmic to nuclear ratio, shownin (C) and a significant increase in the number of nuclei, in (D) arepresented. Scale Bar=100 μm. Abbreviations: Oo (oocyte); GC (granulosacells); A (antrum).

FIG. 3. Using murine multiple follicle culture to phenocopy the humanmenstrual cycle. (A) Late primary and early secondary stage follicles(90 μm to 120 μm) were isolated from mouse ovaries and encapsulated in0.5% alginate in cohorts of 10 follicles per alginate bead. Follicleswere cultured for 14 days (Day-14 to Day 0, in vitro follicular phase).On Day 0, hCG was administered to induce ovulation and follicles werecultured for an additional 15 days (in vitro luteal phase). (i) is theinitial size of follicle (Day-14) (ii) the morphology of follicle onday-10 (iii) is the day follicle was given hCG (Day 0), (iv) folliclescultured in alginate released oocyte (asterisk) in response to hCG.Scale Bar=100 μm. (B) Estradiol and progesterone were measured in spentmedium throughout the culture period. Lighting bolt represents hCGtreatment.

FIG. 4. Individual steroid and peptide hormone profiles in growingsecondary human follicles. Steroid and peptide hormones for each humanfollicle were quantified in the medium throughout the culture period.The culture time on X-axis had been aligned to the time of the hCGaddition (day 0) and cultured for an additional 14-15 days. Theconcentration of (A) estradiol, progesterone, (B) inhibin A, inhibin B,(C) AMH and (D) activin A are reported throughout culture relative tohCG administration. Gray shading represents the in vitro luteal phase,or time post-hCG. Follicles were derived from three donors andindividually cultured.

FIG. 5. Peptide hormone profiles of murine multiple follicle culture.(A) Inhibin A, inhibin B, and (B) AMH were measured throughout theculture period. (B) H&E staining of secondary mouse follicles pre-hCGand post-hCG. Follicles showed morphological signs of luteinization,marked by an increased cytoplasmic to nuclear ratio. Scale Bar=100 μm.

FIG. 6. Murine hCG transcriptional control of steroid and peptidehormones. qRT-PCR was performed on RNA isolated from follicles before(-hCG) and after hCG treatment (1 d, 3 d, and 5 days post-hCG), n=2-3cultures per time point. (A) As reported in vivo, transcripts for thesteroidogenic enzymes Star, Hsd3b1, and Cyp11a1 were significantlyinduced post-hCG. 20αHsd, the enzyme predominately responsible forprogesterone catabolism in the rodent, was upregulated. The enzymesresponsible for androgen and estrogen synthesis, Cyp17a1 and Cyp19a1,respectively, were significantly downregulated post-hCG. (B) Expressionof the gonadotropin receptors genes (Fshr and Lhcgr) also mimicked invivo patterns, with an overall downregulation 3 days post-hCG; (C)Transcription factors known to downregulate inhibin α-subunit expressionin the mouse were induced post-hCG.

FIG. 7. In Vitro Follicle Growth (IVFG) provides a simple and rapidmethod of predicting adverse reproductive outcomes in mammals inresponse to environmental exposures. In IVFG, (A) large numbers ofovarian follicles are collected from pre-pubertal mouse ovaries andplaced individually in wells of a 96-well plate containing chemicals ofinterest. Follicles are cultured and monitored for up to 6 days. (B)Upstream endpoints of IVFG include: (i) follicle survival, (ii) folliclemorphology, (ii) antrum formation, (iv) hormone production, (v) oocytemeiotic competence, and (F) spindle structure. These hallmarks arerobust biomarkers of both endocrine and gamete quality.

FIG. 8. Validation of the IVFG system using two chemotherapeutics knownto have adverse reproductive effects. The effects of increasingconcentrations of CTX and CDDP on follicle (A,B) survival and (C,D)morphology during IVFG are shown. Follicles were treated with CTX andCDDP on day 2 of culture, and follicle survival and morphology wereassessed on days 4 and 6. The arrowheads highlight the antral cavity,and the arrow indicates an oocyte that has been released from thefollicle. Scale bar=100 μm.

FIG. 9. Validation of the IVFG system using two FDA-approvedpharmaceuticals that are not known to have off-target reproductiveeffects. The effects of increasing concentrations of Nalbuphine andRaclopride on follicle (A, B) survival and (C, D) morphology during IVFGare shown. Follicles were treated with Nalbuphine and Raclopride on day2 of culture, and follicle survival and morphology were assessed on days4 and 6. The arrowheads highlight the antral cavity. Scale bar=100 μm.

FIG. 10. CE exposure affects ovarian follicle growth and morphology. Theeffect of increasing concentrations of CE on (A) follicle survival isshown. (B) The survival of follicles, primary ovarian stromal cells, andtransformed HeLa cells following culture in increasing concentrations ofCE was used to calculate the respective LC50 for each cell type. (C) Theeffect of CE exposure on follicle morphology is shown. For theseexperiments, follicles were treated with CE on day 2 of culture, andsurvival and morphology were assessed on days 4 and 6. The arrowheadshighlight the antral cavity. Scale bar=100 μm.

FIG. 11. CE exposure affects follicle differentiation and function. Theeffects of increasing concentrations of CE on (A) antral cavityformation and (B) estradiol production are shown. These experiments wererepeated at least five times with a total of between 51 and 135 follicleexamined per CE dose. Follicles were treated with CE on day 2 ofculture, and antral cavity formation and hormone production wereassessed on days 4 and 6.

FIG. 12. CE exposure affects oocyte meiotic competence and spindlemorphology. The ability of oocytes derived from follicles exposed to CEto progress through the stages of meiotic maturation was (A) scored bylight microscopy and (B) quantified. Cells were classified as germinalvesicle intact (GV), germinal vesicle breakdown/metaphase of meiosis I(GVBD/MI). metaphase of meiosis II (MII), and representative images foreach stage are shown (A). This experiment was repeated at least twiceand a minimum of 25 cells were analyzed in each experimental group. (C)The actin- and microtubule-based cytoskeleton in the resulting MII eggsfrom control and CE-exposed follicles was examined byimmunocytochemistry and confocal microscopy. An optical sectionencompassing the meiotic spindle is shown. Normal meiotic spindles werecharacterized by a bipolar structure with chromosomes tightly aligned onthe metaphase plate (upper panel, control). Abnormal spindles werecharacterized by disrupted tubulin and scattered chromosomes (lowerpanel, 50 ppm CE). (D) The percent of normal spindles observed in theMII eggs derived from follicles exposed to increasing concentrations ofCE was quantifiedArrowheads indicate the polar body. Scale bar=25 μm.

FIG. 13. 3D human fallopian tube in-vitro culture on TRANSWELL membraneinserts maintains tissue architecture and viability. (A). The humanfallopian tube was cut open and the epithelia layer was mechanicallyisolated. The epithelia layer was cut into 2×2 mm pieces and cultured onthe insert membrane. (B) The epithelia layer was treated with low doseof E2 0.1 nM for 14 days and 28 days. H&E staining was used to evaluatethe morphology of human fallopian tube epithelial cells after 14 and 28days in culture compared with uncultured tissue. ERα, PR and OVGP1immunofluorescent staining were performed to characterize the humanfallopian epithelia culture system. Scale bar indicates 100 um size.

FIG. 14. Functional response of human fallopian tube epithelium toculture in the presence of E2 and P4. (A), (B) After 7 days of culturein the indicated treatments, cilia beating frequency was measured usingan Andor spin disk microscope with a 100× objective and a 5 ms exposuretime and 5 ms readout time. (A) Graphs depict a representativemeasurement of cilia beating frequency in cultured fallopian epitheliumfrom 1 patient. (B) Quantification of cilia beating frequency for n=3fallopian endometrium cultures from 3 individual women. (C) Immunoblotof OVGP1 expression in cultured fallopian epithelium after treatment for7 days. Bar graph represents relative band density using α-tubulin asthe loading control for n=4 cultured fallopian endometrium lysates from4 women. (D) Conditioned medium from fallopian epithelium cultures wasused to quantify hIGF1 levels by immunoblot analysis. Bar graphrepresents relative band density using glucose consumption as a loadingcontrol for n=3 conditioned medium samples from fallopian tissuecultures from 3 women. “*” corresponded to statistically significantdifferences between groups. A one-way ANOVA followed by Tukey's multiplecomparisons test was used for statistical analysis, and P<0.05 wasconsidered statistically significant.

FIG. 15. Stepwise exogenous steroid hormone treatment for 28 daysregulates OVGP1 and hIGF1 in human fallopian tube epithelium. (A)Stepwise exogenous E2 and P4 treatments used to mimic the humanmenstrual cycle. (B) Cultured fallopian epithelium was collected on theindicated day and lysate was analyzed for OVGP1 protein expression byimmunoblot. α-tubulin was used as loading control. (C) Conditionedmedium from fallopian endometrial cultures was analyzed by immunoblotfor hIGF1 expression every 7 days. n=3 experiments with conditionedmedia from fallopian tissue cultures from 3 women. Relative density ofeach band was based on E2 0.1 nM treatment group. “*” corresponded tostatistically significant differences between groups. A one-way ANOVAfollowed by Tukey's multiple comparisons test was used for statisticalanalysis, and P<0.05 was considered statistically significant.

FIG. 16. Human fallopian epithelium and murine follicle co-cultureinduces morphological changes and protein secretion. (A) Schematic offollicle and fallopian epithelium co-culture model. Five Secondaryfollicles (150 μm-180 μm) were encapsulated into a single 0.5% alginatebead, which was placed in the bottom of each well. Fallopian epitheliumwas cultured on a 0.4 μm insert membrane, which was placed into the12-well plate containing encapsulated follicles. (B) E2 and P4concentrations from the co-culture medium. The co-cultures weremaintained in growth medium supplemented with 10 mIU/ml recombinanthuman follicle-stimulating hormone (rhFSH) for 7 days. After 7 days, thefollicles were treated with 1.5 IU/ml hCG for 16 hours to induce invitro maturation. The luteinized follicles were then cultured foranother 7 days without rhFSH. “F phase” represents follicular phase, “Lphase” represents luteal phase. (C) Morphology of the fallopianepithelial tissue cultured alone or co-cultured with follicles for 7days and 14 days. Scale bar=100 μm. (D) hIGF1 levels were measured inthe conditioned medium by immunoblot analysis. Bar graph representsrelative band density compared to culture day 5. Three individualexperiments were performed using fallopian tissues from 3 women. “*”Corresponded to statistically significant differences between groups.One-way ANOVA followed by Tukey's multiple comparisons test was used forstatistical analysis, P<0.05 was considered statistically significant.

FIG. 17. Establishment of 3D uterine cultures for 28-days. (A) Primaryendometrial and myometrial cells were cocultured on polystyrene scaffoldand treated with estradiol (E2) and progesterone (P4) in a stepwisefashion; 0.1 nM E2 for 7 days, 1 nM E2 for the next 7 days, followed by1 nM E2+P4 10 nM for 7 days, then 0.1 nM E2+P4 50 nM for 5 days and onlymedia (no hormones) for 2 days for a total culture period of 28 days.Morphology of 3D units are shown by H&E staining. Immunofluorescentstaining of 3D units for vimentin, PR and DAPI revealed presence ofcells and expression of these uterine markers in culture.

FIG. 18. Graphs measuring hormonal regulation of secreted factors withina static 3D uterine culture system.

FIG. 19. Graph of hormone levels used during 28 day culture of uterinecells cultured in decellularized matrix. Myometrial cells were re-seededonto decellurized matrix and cultured for 28 days with the menstrualcycle hormone treatments. H&E staining revealed tissue architecture ofthe recellularized myometrial matrix. DAPI staining revealed thepresence of viable cells within the matrix after 28 days of culture.

FIG. 20. Left, graph of cell survival assay. Right, graph of hormonelevels used during 28 day test of endometrial cell culture response totestosterone levels.

FIG. 21. Images of various cells in the culture subsystems describedherein (top), and a schematic depicting an exemplary flow of secretedfactors from upstream tissues to downstream tissues.

FIG. 22. In vitro support of folliculogenesis, oogenesis, meiosis,ovulation, and embryo development using the 3D culture systems describedherein

FIG. 23. Schematic of translation of static cultures into dynamicmulti-culture system.

FIG. 24. Graph depicting hormone production by encapsulated secondaryfollicles.

FIG. 25. Image demonstrating morphology of follicles recovered afterculture in ovarian microfluidic subsystem.

FIG. 26. Graph demonstrating increased progesterone production fromfollicles in ovarian microfluidic subsystem following hCG treatment.

FIG. 27. Graphs demonstrating the stability of FSH (left) and hCG in theovarian microfluidic subsystem.

FIG. 28. Graphical depiction of the human female 28-day estrous cycle.

FIG. 29. Experimental design for testing the ovarian culture subsystem.

FIG. 30. Drawing of exemplary ovarian culture subsystem.

FIG. 31. Images (top) and graph (bottom) depicting follicular growth instatic culture.

FIG. 32. Images depicting follicular growth in both the follicular phaseand luteal phase supported by an exemplary ovarian culture subsystem.

FIG. 33. Images demonstrating follicle histology following 28 days ofstatic culture and 28 day culture in an exemplary ovarian culturesubsystem.

FIG. 34. Graphs depicting hormone secretion over a 28-day period fromfollicles in an exemplary ovarian culture subsystem.

FIG. 35. Graph comparing estradiol production over a 14-day span fromcells grown in 100 μl static culture and 700 μl microfluidic culture.

FIG. 36. Graphs comparing estradiol production (top left), progesteroneproduction (top right), and follicle diameter over a 14-day span fromcells grown in 100 μl static culture, 700 μl static culture, and 700 μlmicrofluidic culture.

FIG. 37. Graphs comparing hormone expression levels for snorkel and Wspill way media outlet designs.

FIG. 38. Graphs comparing hormone expression levels for culturesubsystems comprising a waste reservoir and collection pore.

FIG. 39. Graphs depicting hormone levels in fresh and 24-hopuraccumulated media over the course of a 28-day cycle in an exemplaryovarian culture subsystem.

FIG. 40. Images (left) and graphs (right) show morphology and hormonesecretion levels of cultured whole ovaries.

FIG. 41. 3D human fimbriae culture system supports secretory andciliated epithelium. Human fimbriae cultures encapsulated in alginatehydrogels retained normal tissue architecture for up to 7 days inculture, as demonstrated by comparative H&E staining. FTE was maintainedas identified by cytokeratin 8 (CK8) staining. Further, both epithelialsubtypes; secretory (PAX8) and ciliated (Ac. Tubulin) Scalebar equals 50μm.

FIG. 42. 3D Fimbriae proliferation in response to estrogen, insulin, andhydrogen peroxide. Proliferation was quantified in the FTE of humanfimbriae treated with 10 nM estradiol (E2), 1×ITS, 1 mM H2O2 or vehicle(Control) for A) 2 and B) 7 days. ITS and H2O2 demonstrated enhancedproliferation after 2 and 7 days in culture. E2 did not alter FTEgrowth. C) Proliferation was determined by 24 hour BrdU labeling(arrows) n=6. Error bars equal mean±SEM. Scalebar equals 50 μm.

FIG. 43. E2 induces PR expression and IL8 secretion from 3D culturedhuman fimbriae. A) Progesterone receptor (PR) expression is limited to aportion of the FTE in control (vehicle treated) samples. Epithelial andstromal expression is induced by treatment with 1 or 10 nM E2 for 7 days(arrows). B) Treatment of fallopian cultures with 10 nM E2 demonstratedinduction of the pro-tumorigenic cytokine IL8 compared to control(vehicle treated). No change was seen in other pro-inflammatorycytokines; C) VEGF-A and D) FGF2. Scalebar equals 50 μm.

FIG. 44. p53 expression is induced in 3D human fimbriae cultures. A) p53expression was evaluated in FTE of 3D human fimbriae samples culturedfor 2 and 7 days with vehicle (control), 10 nM E2, 1×ITS, or 1 mM H2O2.p53 expression was not induced by an specific treatment. However, p53expression did appear to be induced by culturing alone. B) p53signatures were not identified in uncultured samples, but were noted inex vivo cultured fimbriae (arrow) primarily in secretory epithelium(PAX8), but not always coincidental with DNA damage (pH2AX) althoughdamage was seen in stromal cells (arrow). Scalebar equals 50 μm.NS=non-specific staining.

FIG. 45. Cell viability following a step hormone treatment for 28-days.(A) Primary endocervical cells were treated with estradiol (E2) andprogesterone (P4) in a stepwise fashion; 0.1 nM E2 for 7 days, 1 nM E2for the next 7 days, followed by 1 nM E2+P4 10 nM for 7 days, then 0.1nM E2+P4 50 nM for 5 days and only media (no hormones) for 2 days for atotal culture period of 28 days. (B) Endocervical cells were infectedwith 2 μl of pAD-eGFP-RLC (Ad-GFP) at day-0 of hormonal treatment. Bythe end of 28-day treatment regimen, viable endocervical cells werepresent, as visualized with fluorescence in GFP infected cells. Thebackground was shown in red. The adherent uninfected cells can beobserved by bright-field microscopy, in which reduced light reflectionis a result of increased cell density observed specially in the presenceE2+P4. Magnification 40×. (C) Cell viability was measured by Alamar bluestaining. Data points on the graph represent the means±SD of fourindependent experiments. Statistical comparisons (student t-test)between E2 and E2+P4 were performed (p<0.05).

FIG. 46. Establishment of 3D cultures of the human endocervix. Theendocervix tissue was obtained post-surgery and tissue was enzymaticallydigested. Cells were cultured on 2D culture plates and expanded. Cellswere trypsinized and seeded at 2×106 cells/well onto the polystyrenescaffold membrane. Cells were cultured for 28-days in the presence ofsteroid hormones. The 3D units were fixed and processed for hematoxylinand eosin (H&E) staining.

FIG. 47. Expression of endocervical cell markers. Cells in thepolystyrene scaffold were fixed, processed and (A) stained withhematoxylin and eosin (H&E) at day-14 (E2-only) and day-28 (Control andE2+P4). (A) Immunohistochemical staining for estrogen receptor (ER),progesterone receptor (PR), (B) pan cytokeratin, vimentin and Ki-67 wasdone. (C) MUC16 levels were detected by IHC staining, neutral mucins byPeriodic Acid Schiff (PAS) staining and acidic mucins by Alcian blue(AB) at day-14 (E2) and day-28 (Control and E2+P4) in 3D endocervicalcells. The PAS stain revealed the presence of goblet cells (arrow).Alcian blue staining detected acidic mucins (arrow). Figures arerepresentative of at least three independent experiments. Scale barsrepresent 25 μm (inset) and 50 μm. (D) Cells were treated with 100 nMRU486. Levels of MUC16 released into the culture media were measured byELISA. Data are means±SD from three patient samples.

FIG. 48. Hormonal regulation of IL-1β and LIF. Endocervical cells in 3Dwere treated with the step hormone treatment. (A) A 45-plex Luminexassay was performed, using media collected at D14, D26 and D28. Data arepresented as fold changes from vehicle controls (n≧4 patient samples). *denotes p<0.05. (B) IL-1β and LIF levels were validated by ELISA. RU486was added to the cells at D21. Data are mean±SD from 3 patient samples.Statistical comparisons among all treatments were performed (p<0.05).

FIG. 49. The four layers of the ectocervix.

FIG. 50. The ectocervix changes throughout the menstrual cycle inresponse to ovarian hormones, our model should recapitulate thesechanges by becoming glycogenated and differentiating into a maturestratified squamous epithelium in response to estrogen. As estrogenlevels drop off, the most superficial layer of cells is shed in aprocess known as desquamation. Additionally, progesterone receptorexpression should increase in response to estrogen, whereas thesecretion of antimicrobial peptides will decrease in response toestrogen. This model will be used to determine hormonally regulatedmechanisms involved in barrier function, and to discover additionalhormonally regulated secreted factors, such as cytokines and chemokines.

FIG. 51. Establishment of static 3d cultures of primary epithelial cellsusing collagen hydrogels and J2-3T3 stromal fibroblasts at a liquid-airinterface.

FIG. 52. The day 7 models showed the basal and parabasal layers hadformed, with the more superficial layers beginning to form in the ROCKinhibitor treated cells. By day 14, all four layers were apparent inboth the primary and ROCK inhibitor treated cells. Glycogenation wasapparent in the intermediate and superficial layers, shown by largewhite spaces in the cytoplasm, a sign of normal maturation anddevelopment of the stratified squamous epithelium.

FIG. 53. Cultures were harvested after 7 and 14 days forimmunohistochemical analysis of ectocervix and differentiation markers.All differentiation markers were detected as early as 7 days in culture(A-D), and mimicked expression patterns of normal ectocervix tissue(NEC) (I-L) by day 14 (E-H). CK13, expressed in non-keratinizingstratified squamous epithelium, was found in the parabasal, intermediateand superficial layers (A, E, I). CK14, a stratified squamous basallayer marker, was present in the models and NEC (B, F, J). P63 drivesdifferentiation in the ectocervix and was expressed in the basal andparabasal layers (C, G, K). MUC4 plays a role in lubrication, hydrationand barrier protection in the ectocervix and was expressed throughoutthe epithelium in both the models and NEC (D, H, L). Scale bar=50 um.

DEFINITIONS

As used herein the terms “3D culture,” “3D cell culture,” and 3D tissueculture” refer to artificial environments in which biological cells arepermitted to grow and/or interact surroundings in all three dimensions.This is in contrast to traditional 2D culture in which cells are grownin a monolayer on a flat, two-dimensional surface, such as a petri dish.Environments for 3D culture include extracellular matrices, 3Dscaffolds, polymer (e.g., alginate) encapsulation, bioreactors (e.g.,rotating bioreactors), microcarriers, magnetic levitation systems,hanging drop, magnetic 3D bioprinting, etc.

DETAILED DESCRIPTION

The present invention relates generally to a three-dimensional cell andtissue culture system for the female reproductive tract. In particularprovided herein the system includes individual female reproductivecultures integrated using a microfluidic microphysiologic system. Insome embodiments, the present invention provides ex-vivo femalereproductive tract integration in a three dimensional (3D)microphysiologic system. In particular embodiments, provided herein areex vivo female reproductive tracts constructed using individual 3Dtissue culture subsystems integrated into a microfluidicmicrophysiologic system (e.g., FemKube). In some embodiments, the systemincludes individual 3D cultures of one or more of ovarian follicles(e.g., OvaryKube), fallopian tube (e.g., TubeKube), uterus (e.g.,UteroKube), and cervix (e.g., CerviKube). In some embodiments, thecervix is divided into separate endocervix and ectocervix cultures. Insome embodiments, each tissue remains viable for at least one menstrualcycle (e.g., 28 days for humans) and responds to hormonal fluctuationsthat mimic the menstrual cycle (e.g., human menstrual cycle).

3D culture systems for the female reproductive tract do not exist usinghuman cells that are relevant and/or useful of the time scale relevantto long term physiologic processes (e.g., menstrual cycle). In someembodiments, the systems provided herein utilize hormones secreted by 3Dcultured ovarian follicles to stimulate other downstream reproductivetissues rather than adding exogenous hormones to the cell culture media.In some embodiments, a series of 3D culture subsystems are in fluidcommunication such that each subsequent subsystem reacts to the hormonesand other factors released by the upstream subsystems (e.g., thesubsystem immediately upstream, all subsystems upstream, etc.). In someembodiments, flow between subsystems is regulated (e.g., bymicrofluidics) in such a manner as to allow the appropriate level ofsecreted hormones and other factors to pass one or more subsequentsubsystems. In some embodiments, the relative volume of subsystems isselected to allow the appropriate level of secreted hormones and otherfactors to pass one or more subsequent subsystems.

In some embodiments, a system comprises multiple culture subsystems influid communication (e.g., bidirectional, unidirectional, continuous,with mixing, without mixing, etc.). For example, unidirectional fluidcommunication allows downstream tissues to respond to factors secretedby upstream tissues in real time to establish a system that mimics thedynamic female reproductive tract ex vivo (See, e.g., FIG. 21). In someembodiments, subsystems are connected in series (e.g., as depicted inFIG. 21). In other embodiments, two or more subsystems are connected inparallel (e.g., downstream and upstream from the same subsystems). Insome embodiments, two or more subsystems are connected in semi-parallel(e.g., downstream or upstream from the same subsystems, but in serieswith one or more other subsystems).

In some embodiments, dynamic culture allows for the transfer of factorsfrom one culture subsystem to another, thereby recapitulating thetransfer of factors within complex body systems. Multiple staticcultures in which the necessary factors are added by a user or automatedsystem are translated into dynamic systems in which the cells/tissuesare able to communicate as they do in vivo, with real-time transfer(e.g., downstream delivery) of hormones and other factors betweentissues (FIG. 23).

A microphysiologic system, containing living cell tissue constructsinterconnected by microfluidic channels is described, for example, inU.S. Pat. No. 7,288,405; herein incorporated by reference in itsentirety. In some embodiments, the systems described herein permit cellsto be maintained in vitro, under conditions similar to those found invivo. Parameters accurately simulated by the system include, forexample, interactions between cells, liquid residence time, liquid tocell ratios, relative size, metabolism by cells, shear stress, and thelike. In some embodiments, systems mimic the natural state of cells, thepredictive value of assays performed therewith. In some embodiments, amicrofluidic network of channels connect segregated, discrete chambers.In some embodiments, chamber geometry and connectivity is designed toprovide cellular interactions, liquid flow, and liquid residenceparameters that correlate with those found for the corresponding cells,tissues, or organs in vivo.

In some embodiments, microphysiologic systems find use in, for example,pharmaceutical and toxicology testing for effects on the reproductivetract (e.g., without using animals), contraception and/or hormonetesting, reproductive biology studies of normal and/or diseased tissues.Systems described herein have numerous advantages, including but notlimited to, the use of primary human tissues rather than animal tissuesor cell lines, they maintain viability and/or hormonal response over thelength of the normal (human) menstrual cycle, they provide the abilityto use 3D cultured ovarian follicles as hormone source for other 3Dcultured tissues to mimic human menstrual cycle.

In some embodiments, a system comprises multiple individual femalereproductive 3D cultures (e.g., two, three, four, five, six, or more).In some embodiments, the separate 3D cultures (e.g., subsystems) areintegrated using a microfluidic microphysiologic system. The individualcultures are, for example, ovarian follicles, fallopian tube, uterus,and cervix (e.g., endocervix and ectocervix. In some embodiments, theovarian culture subsystem uses either human or murine folliclesencapsulated in polymer (e.g., collagen, alginate, poly (octanediolcitrate), etc.). In some embodiments, the encapsulated follicles remainviable long term (e.g., 28+ days). In some embodiments, the encapsulatedfollicles respond to follicle stimulating hormone (FSH) and humanchorionic gonadotropin (hCG) in-vitro follicle maturation by producingestrogen and progesterone in a pattern that mimics the human menstrualcycle. In some embodiments, the fallopian tube subsystem uses humanfallopian epithelium tissue pieces grown, for example, on TRANSWELLinserts. Tissue remains viable for 28 or more days and maintains bothsecretory and ciliated epithelium cell phenotypes. In some embodiments,the fallopian epithelium functionally responds to estrogen andprogesterone using secreted factors and cilia beating as markers. Theuterine subsystem is comprised of human endometrial epithelial,endometrial stromal, and myometrial cells. In some embodiments, theendometrial epithelial and stromal cells are isolated separately andcombined for culture on TRANSWELL inserts, while myometrial smoothmuscle cells are cultured on a separate TRANSWELL insert. In someembodiments, the endometrial and myometrial inserts are cultured in thesame tissue culture well in a common media. Uterine cultures are viablefor at least 28 days. In some embodiments, cervical cultures arecomprised of primary human endocervical epithelial and stromal cellsgrown on the same TRANSWELL insert. Endocervix remains viable for atleast 28 days and responds to estrogen and progesterone mimicking thehuman menstrual cycle.

Provided herein is a multi-component culture system comprised ofmultiple culture subsystems, wherein each of the culture subsystems isin fluid communication (e.g., bidirectional, unidirectional, continuous,with mixing, without mixing, etc.) with at least one of the othersubsystems. In some embodiments, appropriate culture conditions areprovided for each culture subsystem (e.g., media, temperature, etc.).

In some embodiments, culture media is changed regularly (e.g., hourly,four-times daily, twice daily, daily, etc.). In some embodiments,culture media is continuously replenished. In some embodiments, the flowof media, reagents, and other chemicals (e.g., hormones) between thesubsystems renders one or more subsystems and/or the entire systemself-replenishing. In some embodiments, culture is carried out atbetween 18° C. and 40° C. (e.g., 18° C., 19° C., 20° C., 21° C., 22° C.,23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C.,32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C.or ranges therein). In some embodiments, culture is carried out at roomtemperature (e.g., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24°C., or ranges therein). In some embodiments, culture is carried out athuman physiologic temperature (e.g., about 37° C.). In some embodiments,reagents used in culture media are sterilized. In some embodiments,devices (e.g., chambers, vessels, bottles, flasks, tubes, etc.) used inculturing are sterilized.

In some embodiments, appropriate devices are selected for containingcells and media in the various culturing subsystems. In someembodiments, Transwell plates or other permeable supports are provided.Transwell cell culture chambers, or Transwell plates (e.g., availablefrom Costar Corp., Cambridge, Md., USA) are an example of a multichamberculture device in which media, nutrients, hormones, reagents, etc. maypass between the chambers, but cells may not. Each chamber of aTranswell plate comprises a flat-bottomed, open-topped, lowercompartment with impermeable bottom and sides, and an open-topped, uppercompartment with a microporous membrane which forms the bottom of theupper compartment. This assembly is typically covered by a removablelid. In use, cells (e.g., a first type of cells) are placed on the uppersurface of the microporous membrane within the upper compartment. Theupper compartment is inserted into the lower compartment. Due to thepermeability of the membrane, media, nutrients, factors, etc. are ableto traverse the membrane. Cells may also be placed in the upper chamber(e.g., a second type of cells). Other multi-chamber culture systems(e.g., two chambers, three chambers, four chambers, or more) may finduse in embodiments described herein. Divisions between chambers may bepermeable, semipermeable (e.g., with a particular molecular weightcutoff (e.g., permeable to small molecules, but not proteins), orimpermeable.

In some embodiments, ovarian follicle culture media comprises one ormore (e.g., all) of: alpha-MEM, DMEM F12, BSA, Fetuin, Insulin,Transferrin, Selenium, and FSH. In some embodiments, ovarian follicleculture media comprises one or more (e.g., all) of: alpha-MEM (e.g.40-60%, 45-55%, about 50%, etc.), DMEM F12 (e.g. 40-60%, 45-55%, about50%, etc.), BSA (1-5 mg/ml, 2-4 mg/ml, about 3 mg/ml, etc.), Fetuin(0.1-1 mg/ml, 0.3-0.7 mg/ml, about 0.5 mg/ml), Insulin (1-10 mg/ml, 3-7mg/ml, about 5 mg/ml), Transferrin (1-10 mg/ml, 3-7 mg/ml, about 5mg/ml), Selenium (1-10 ng/ml, 3-7 ng/ml, about 5 ng/ml), and FSH (e.g.,5-15 IU/ml, 8-12 IU/ml, about 10 IU/ml). In some embodiments, ovarianfollicle culture media comprises one or more (e.g., all) of: 50%alpha-MEM, 50% DMEM F12, 3 mg/mL BSA, 0.5 mg/mL Fetuin, Insulin (5ug/mL), Transferrin (5 ug/mL), Selenium (5 ng/mL), 10 IU/mL FSH. In someembodiments, during hCG treatment to trigger the luteal phase of thein-vitro menstrual cycle, the media is changed. In some embodiments, theluteal phase media comprises one or more (e.g., all) of: alpha-MEM, FBS,EGF, hCG, and FSH. In some embodiments, the luteal phase media comprisesone or more of: alpha-MEM (e.g., 90-100%, about 100%, etc.), FBS (1-20%,5-15%, 8-12%, about 10%, etc.), EGF (1-10 ng/ml, 3-7 ng/ml, about 5ng/ml, etc.), hCG (0.5-2.5 IU/ml, 1.0-2.0 IU/ml, about 1.5 IU/ml, etc.),and FSH (5-15 IU/ml, 8-12 IU/ml, about 10 IU/ml, etc.). In someembodiments, the luteal phase media comprises one or more (e.g., all)of: 100% alpha-MEM, 10% FBS, EGF (5 ng/mL), hCG (1.5 IU/mL), FSH (10IU/mL). In some embodiments, following hCG treatment (e.g., for 4-28hours, 10-22 hours, 14-18 hours, about 16 hours), the second media isreplaced with the first media formulation but with reduced FSH (e.g.,without FSH).

In some embodiments, fallopian culture media comprises one or more(e.g., all) of: alpha MEM, BSA, Fetuin, Insulin, Transferrin, andSelenium. In some embodiments, fallopian culture media comprises one ormore (e.g., all) of: alpha MEM, BSA (e.g., 1-5 mg/ml, 2-4 mg/ml, about 3mg/ml, etc.), Fetuin (e.g., 0.1-1.0 mg/ml, 0.3-0.7 mg/ml, about 0.5mg/mL, etc.), Insulin (e.g., 1-10 mg/ml, 3-7 mg/ml, about 5 mg/mL,etc.), Transferrin (e.g., 1-10 mg/ml, 3-7 mg/ml, about 5 mg/mL, etc.),Selenium (e.g., 1-10 ng/ml, 3-7 ng/ml, about 5 ng/mL, etc.), etc. Insome embodiments, fallopian culture media comprises one or more (e.g.,all) of: alpha MEM, BSA (e.g., 3 mg/ml), Fetuin (e.g., 0.5 mg/mL),Insulin (e.g., 5 mg/mL), Transferrin (e.g., 5 mg/mL), Selenium (e.g., 5ng/mL), etc.

In some embodiments, uterine culture media comprises one or more (e.g.,all) of: DMEM F12, Insulin, Transferrin, Selenium, pen-strep, strippedFBS, etc. In some embodiments, uterine culture media comprises one ormore (e.g., all) of: DMEM F12, Insulin (e.g., 1-10 μg-ml, 3-7 μg/ml,about 5 μg/ml, etc.), Transferrin (e.g., 1-10 μg-ml, 3-7 μg/ml, about 5μg/ml, etc.), Selenium (e.g., 1-10 ng/ml, 3-7 ng/ml, about 5 ng/mL,etc.), pen-strep (0.1-10%, 0.5-5%, about 1%, etc.), stripped FBS (e.g.,1-10%, 3-7%, about 5%, etc.), etc. In some embodiments, uterine culturemedia comprises one or more (e.g., all) of: DMEM F12, Insulin (5 ug/mL),Transferrin (5 ug/mL), Selenium (5 ng/mL), 1% pen-strep, 5% strippedFBS, etc.

In some embodiments, endocervix culture media comprises one or more(e.g., all) of: KBM (Keratinocyte Basal Medium), Insulin, Transferrin,EGF, Gentamicin-amphotericin-B, Bovine Pituitary Extract (BPE),Ephinephrine, hydrocortisone, etc. In some embodiments, endocervixculture media comprises one or more (e.g., all) of: KBM (KeratinocyteBasal Medium), Insulin (e.g., 1-10 μg/ml, 3-7 μg/ml, about 5 μg/ml,etc.), Transferrin (e.g., 1-20 μg/ml, 5-15 μg/ml, about 10 μg/ml, etc.),EGF (e.g., 0.025-1 μg/ml, 0.05-0.25 ng/ml, about 0.125 ng/ml, etc.),Gentamicin-amphotericin-B (e.g., 0.01-1%, 0.05-0.5%, about 0.1%, etc.),Bovine Pituitary Extract (BPE) (e.g., 0.1-1%, 0.2-0.6%, about 0.4%,etc.), Ephinephrine (e.g., 0.2-0.5 μg/ml, 0.3-0.45 μg/ml, about 0.39ug/ml, etc.), hydrocortisone (e.g., 0.1-0.5 μg/ml, 0.2-0.4 μg/ml, about0.33 μg/ml, etc.). In some embodiments, endocervix culture mediacomprises one or more (e.g., all) of: KBM (Keratinocyte Basal Medium),Insulin (5 ug/mL), Transferrin (10 ug/mL), EGF (0.125 ng/mL),Gentamicin-amphotericin-B (0.1%), Bovine Pituitary Extract (BPE) (0.4%),Ephinephrine (0.39 ug/mL), and hydrocortisone (0.33 ug/mL).

In some embodiments, endocervix culture media comprises one or more(e.g., all) of: adenine, human recombinant, human apo-transferin,triiodothyronine, DMEM HG, DMEM:F-12, gentamicin, amphotericin, 4 mmL-Glut, hydrocortisone, and cholera toxin in amounts plus or minus 50%,40%, 30%, 20%, or 10% of the concentrations in Example 8.

In some embodiments, two or more subsystems (e.g., all) utilize the samemedia (e.g., universal media). In some embodiments, universal mediacomprises one or more of the aforementioned components.

In some embodiments, a microphysiologic system is provided comprisingone or more (e.g., two or more, 3, 4, 5, 6, or more) culture subsystemsin fluid communication via microfluidics. In some embodiments, thepresent invention is not limited by the identity, manufacturer (e.g.,Draper Laboratory, Quiatech, etc.), or type of microfluidic system.

In some embodiments, the present invention provides compositions andmethods for a female reproductive tract organ system capable ofmimicking the female menstrual cycle (See, e.g., Example 1).

In some embodiments the present invention provides a female reproductivetract organ system capable of assessing the effects of environmentalcontaminants on female reproduction (See, e.g., Example 2).

In some embodiments the present invention is capable of assessing thebiological function of a single organ in the organ system including itsresponse to the female menstrual cycle (See, e.g., Example 3).

In some embodiments, a microphysiologic system comprises a subsystemthat mimics the behavior of the human uterus. Experiments were conductedduring development of embodiments of the present invention to producesuch a subsystem (See, e.g., Example 4).

In some embodiments, a microphysiologic system comprises a subsystemthat mimics the behavior of the human ovaries. Experiments wereconducted during development of embodiments of the present invention toproduce such a subsystem (See, e.g., Example 5).

In some embodiments, a microphysiologic system comprises a subsystemthat mimics the behavior of the human fallopian tubes. Experiments wereconducted during development of embodiments of the present invention toproduce such a subsystem (See, e.g., Example 6).

In some embodiments, a microphysiologic system comprises a subsystemthat mimics the behavior of the human endocervix. Experiments wereconducted during development of embodiments of the present invention toproduce such a subsystem (See, e.g., Example 7).

In some embodiments, a microphysiologic system comprises a subsystemthat mimics the behavior of the human ectocervix. Experiments wereconducted during development of embodiments of the present invention toproduce such a subsystem (See, e.g., Example 8).

Although some embodiments are described as pertaining to the humanfemale reproductive tract, the present invention is not so limited, andmay find use with non-human animals as well (e.g., other mammals,non-human primates, rodents, canines, felines, bovines, equines,porcines, etc.).

EXPERIMENTAL Example 1 Engineering Rodent and Human Hormonal MenstrualCycles in an Artificial Ovary

The menstrual cycle in humans and estrous cycle in rodents represent aseries of hormone and tissue changes that support female fertility andendocrine health. To easily study hormonal changes over the menstrualcycle, we developed an encapsulated three dimensional (3D) human andmurine in vitro follicle growth (eIVFG) system that creates a physicalenvironment that supports both follicle development, includinglutenization, and hormone secretion. The hormones from eIVFG-culturedhuman and mouse follicles were profiled, and the genes involved in thefollicular-luteal transition in mouse follicle culture werecharacterized. Furthermore, mouse follicles were able to secrete steroidhormones in a pattern that mimics the human 28 day cycle. The 3D eIVFGculture system reliably phenocopies in vivo follicle hormone productionand gene expression profiles, providing new opportunities to probemolecular mechanisms and the effects of iatrogenic insults on folliclefunction in the cycling female.

One purpose of the female reproductive axis is to produce, through thecomplex process of folliculogenesis, a terminally differentiated haploidegg that can be fertilized by sperm to produce a viable embryo (E. A.McGee, A. J. Hsueh, Initial and cyclic recruitment of ovarian follicles.Endocrine reviews 21, 200 (April, 2000); herein incorporated byreference in its entirety). The central organ of the female reproductiveaxis is the ovary and the basic unit of the ovary is the follicle, whichis composed of a germ cell surrounded by somatic cells, (granulosa andtheca cells). Large follicles are visible on the surface of the ovary asfluid filled, blister-like structures containing a single oocyte thatwill be released from the ovary into the oviduct, or fallopian tube, atovulation. During human development, one million follicle-enclosedoocytes arrest in meiotic prophase, a resting state that can last foryears or decades before beginning a program of development that leads toa mature, fertilizable egg. Once the follicle is selected to resumegrowth, the somatic cells produce and release hormones, like the steroidhormones estrogen and progesterone, with carefully orchestrated timingto control oocyte development, menses, and implantation while alsocoordinating hormonally responsive signaling pathways throughout thebody. Therefore, follicle produced hormonal fluctuations are aubiquitous part of female physiology and have important effects on thefunction of non-reproductive organs and therapeutic outcomes.

Avenues for studying in vivo human follicle development and theinfluence of hormonal fluctuations during the menstrual cycle on humanphysiology are limited. Therefore, in vitro models that recapitulate the28-day human menstrual cycle using three dimensional (3D) follicleculture are required to completely understand not only reproductivefunction, but also whole body health. The recent development of in vitrofollicle culture by our group has made it possible, for the first time,to assess hormone production by human and mouse follicles at variousstages of growth (M. Xu et al., Encapsulated three-dimensional culturesupports development of nonhuman primate secondary follicles. Biology ofreproduction 81, 587 (September, 2009); M. Xu, A. Banc, T. K. Woodruff,L. D. Shea, Secondary follicle growth and oocyte maturation by culturein alginate hydrogel following cryopreservation of the ovary orindividual follicles. Biotechnology and bioengineering 103, 378 (Jun. 1,2009); herein incorporated by reference in their entireties).Experiments were conducted during development of embodiments of thepresent invention to build a new model that recapitulates a complete invivo follicle hormone production cycle in vitro in order to advance ourunderstanding of follicle biology and the environment in which thegamete develops. Application of this model includes integration of 3Dmaturing follicles into a microphysiologic system to study the effectsof ovarian hormones on the female reproductive tract andnon-reproductive tissues.

It has been shown that an alginate-based encapsulated in vitro folliclegrowth (eIVFG) system supports coordinated folliculogenesis andoogenesis in follicles from all mammalian species examined to date (M.Xu et al., In vitro grown human ovarian follicles from cancer patientssupport oocyte growth. Human reproduction 24, 2531 (October, 2009); M.Xu et al., Encapsulated three-dimensional culture supports developmentof nonhuman primate secondary follicles. Biology of reproduction 81, 587(September, 2009); M. Xu, A. Banc, T. K. Woodruff, L. D. Shea, Secondaryfollicle growth and oocyte maturation by culture in alginate hydrogelfollowing cryopreservation of the ovary or individual follicles.Biotechnology and bioengineering 103, 378 (Jun. 1, 2009); M. Xu et al.,In vitro oocyte maturation and preantral follicle culture from theluteal-phase baboon ovary produce mature oocytes. Biology ofreproduction 84, 689 (April, 2011). herein incorporated by reference intheir entireties). In this study, we performed a comprehensive analysisof the hormones produced by human preantral follicles during a longereIVFG cycle that included a FSH-driven follicular phase, and ovulationstimulus and an in vitro luteal phase. Secondary stage human follicles(120 μm to 260 μm diameter) were isolated from ovarian tissue donatedfor research under IRB approval and following informed consent.Follicles were encapsulated in 0.3% alginate, and levels of estradiol,progesterone, inhibin A, inhibin B, AMH, and activin A were measured inthe spent culture media throughout culture (FIG. 1). The in vitrofollicular phase of development was tracked for each individual follicleby measuring estradiol, and once these levels plateaued at approximately15 days of culture, luteinization was triggered by human chorionicgonadotropin (hCG) administration for 36 hours. Luteninizationrepresents the terminal differentiation of estrogen-producing granulosacells into progesterone-producing luteal cells. The in vitro lutealphase was monitored for an additional 15 days post-hCG, resulting intotal cultures times that ranged from 40 to 65 days.

Although the absolute levels of steroid and peptide hormones produced byindividual follicles varied (FIG. 4), the overall patterns were similarto each other and to hormone cycle measured in vivo (N. A. Klein et al.,Age-related analysis of inhibin A, inhibin B, and activin a relative tothe intercycle monotropic follicle-stimulating hormone rise in normalovulatory women. The Journal of clinical endocrinology and metabolism89, 2977 (June, 2004); J. MacNaughton, M. Banah, P. McCloud, J. Hee, H.Burger, Age related changes in follicle stimulating hormone, luteinizinghormone, oestradiol and immunoreactive inhibin in women of reproductiveage. Clinical endocrinology 36, 339 (April, 1992); C. K. Welt, Y. L.Pagan, P. C. Smith, K. B. Rado, J. E. Hall, Control offollicle-stimulating hormone by estradiol and the inhibins: criticalrole of estradiol at the hypothalamus during the luteal-folliculartransition. The Journal of clinical endocrinology and metabolism 88,1766 (April, 2003); herein incorporated by reference in theirentireties) Inhibin B was the predominant inhibin produced by smallantral follicles, rising in the early to mid-follicular phase anddeclining in the later follicular phase. Inhibin A and estradiol levelsincreased and reached maximal levels as the follicles reached maturityduring the follicular phase. Post-hCG, progesterone levels increased andthere was a trend towards higher activin A, as is found in human serum(S. Muttukrishna, P. A. Fowler, L. George, N. P. Groome, P. G. Knight,Changes in peripheral serum levels of total activin A during the humanmenstrual cycle and pregnancy. The Journal of clinical endocrinology andmetabolism 81, 3328 (September, 1996); herein incorporated by referencein its entirety) Inhibin A levels dropped transiently with hCGtreatment, but then rose and remained elevated throughout the lutealphase. In contrast, inhibin B levels remained low during the lutealphase. The discordance of inhibin A and inhibin B during the lutealphase is unique to primates and is replicated here (N. A. Klein et al.,Age-related analysis of inhibin A, inhibin B, and activin a relative tothe intercycle monotropic follicle-stimulating hormone rise in normalovulatory women. The Journal of clinical endocrinology and metabolism89, 2977 (June, 2004); C. K. Welt, J. E. Hall, J. M. Adams, A. E.Taylor, Relationship of estradiol and inhibin to thefollicle-stimulating hormone variability in hypergonadotropichypogonadism or premature ovarian failure. The Journal of clinicalendocrinology and metabolism 90, 826 (February, 2005); hereinincorporated by reference in their entireties). AMH, a hormone usedclinically as a surrogate marker of small follicles, reached its peak inthe early follicular phase and dropped before mid-follicular phase.Serum AMH levels in women remain constant during the menstrual cycle,reflecting the fact that AMH is made by a fairly constant recruitment ofimmature follicles into the growing pool C. Weenen et al.,Anti-Mullerian hormone expression pattern in the human ovary: potentialimplications for initial and cyclic follicle recruitment. Molecularhuman reproduction 10, 77 (February, 2004); A. L. Durlinger, J. A.Visser, A. P. Themmen, Regulation of ovarian function: the role ofanti-Mullerian hormone. Reproduction 124, 601 (November, 2002); hereinincorporated by reference in their entireties). The culture ofindividual human follicles shows that on a per follicle basis, AMH ismade only through the time of antrum cavity formation, a new discoverythat provides opportunity for study of signaling pathways that controlthis marker of a developing follicle.

In addition to changes in hormone secretion, ovarian follicles underwentmorphological changes representative of in vitro maturation.Specifically, ovarian follicles completed a growth phase (FIG. 2A),followed by luteinization post-hCG. The growth phase coincided with theexpected timing of the follicular phase, while post-hCG, histologicalanalysis showed the characteristic granulosa cell hypertrophy associatedwith luteinization (FIG. 2B-D). These results indicate that theendocrinology of the human follicle is faithfully recapitulated in theeIVFG system.

Although this culture technique provides a robust method to studyfollicular and luteal endocrinology, access to human follicles islimited. Thus, experiments were conducted during development ofembodiments of the present invention to phenocopy the hormone productionof the human menstrual cycle using eIVFG cultured mouse follicles. Lateprimary stage follicles were isolated from 12-day old mouse ovaries andencapsulated in 0.5% alginate in cohorts of 10 follicles. Follicles werecultured for 14 days (in vitro follicular phase) upon which hCG wasadministered to induce ovulation followed by an additional 15 days ofculture (in vitro luteal phase) (FIG. 3A). The patterns of estradiol,progesterone, inhibin B and AMH production during this 29-day culturemimicked a complete human menstrual cycle with both follicular andluteal phases (FIG. 3B and FIG. 5). The phenocopied human menstrualcycle was capitalized by co-culturing mouse follicles with 3D humanfallopian tube epithelia. Hormones produced by mouse folliclesstimulated human fallopian epithelia cilia beating and expression ofoviductal proteins.

A difference between human and mouse is the luteal phase secretionpattern of the peptide hormone inhibin A. In the primate, inhibin A isexpressed post-LH and augments progesterone production (W. Ge, R. E.Peter, Activin-like peptides in somatotrophs and activin stimulation ofgrowth hormone release in goldfish. General and comparativeendocrinology 95, 213 (August, 1994); herein incorporated by referencein its entirety) and suppresses FSH T. K. Woodruff et al., Inhibin A andinhibin B are inversely correlated to follicle-stimulating hormone, yetare discordant during the follicular phase of the rat estrous cycle, andinhibin A is expressed in a sexually dimorphic manner. Endocrinology137, 5463 (December, 1996);

K. I. Parker, D. M. Robertson, N. P. Groome, K. L. Macmillan, Plasmaconcentrations of inhibin a and follicle-stimulating hormone differbetween cows with two or three waves of ovarian follicular developmentin a single estrous cycle. Biology of reproduction 68, 822 (March,2003); T. A. Molskness, T. K. Woodruff, D. L. Hess, K. D. Dahl, R. L.Stouffer, Recombinant human inhibin-A administered early in themenstrual cycle alters concurrent pituitary and follicular, plussubsequent luteal, function in rhesus monkeys. The Journal of clinicalendocrinology and metabolism 81, 4002 (November, 1996); B. S. Houmard etal., Age-related analysis of inhibin A and B relative to the intercyclemonotropic FSH rise in normal ovulatory women. Annales d'endocrinologie64, 86 (April, 2003); herein incorporated by reference in theirentireties). In the mouse, the inhibin subunits (α and β_(A) and β_(B))are transcriptionally repressed by the NR4A and C/EBPβ transcriptionfactors (A. D. Burkart, A. Mukherjee, E. Sterneck, P. F. Johnson, K. E.Mayo, Repression of the inhibin alpha-subunit gene by the transcriptionfactor CCAAT/enhancer-binding protein-beta. Endocrinology 146, 1909(April, 2005); herein incorporated by reference in its entirety). Toexplore species-specific inhibin A regulation in the 29-day mousefollicle culture system, inhibin A secretion and gene expression wasexamined. As predicted by the mouse hormone patterns measured in vivo,in vitro inhibin A fell at the time of hCG. This result indicates thatthe species-specific mechanisms governing mouse and human inhibinsubunit expression are retained in the eIVFG setting although thehormone secretion pattern from mouse follicles mimicked a humanmenstrual cycle. Given the limited amount of human tissue, examinationof gene expression profiles of NR4A and C/EBPβ was not possible.However, using the mouse system, we quantified the expression of thesetranscription factors during the in vitro mouse luteal phase of culturemouse follicles (FIG. S3). Nr4a1-3 and C/EBPβ were upregulated post-hCG,providing evidence that cultured rodent follicles were following thenormal murine pattern of gene regulation. In addition, expression wasexamined of steroidogenic enzymes (Star, Hsd3β1, Cyp11α1 and Akr1c18)known to be upregulated post-hCG in the mouse as well as the follicularenzymes responsible for estrogen synthesis (Cyp17α1 and Cyp19α1).Expression of these enzyme systems phenocopied patterns observed in vivo(Ronen-Fuhrmann et al., Spatio-temporal expression patterns ofsteroidogenic acute regulatory protein (StAR) during folliculardevelopment in the rat ovary. Endocrinology 139, 303 (January, 1998); L.Hedin, R. J. Rodgers, E. R. Simpson, J. S. Richards, Changes in contentof cytochrome P450(17)alpha, cytochrome P450scc, and3-hydroxy-3-methylglutaryl CoA reductase in developing rat ovarianfollicles and corpora lutea: correlation with theca cellsteroidogenesis. Biology of reproduction 37, 211 (August, 1987); M. F.Hay, R. M. Moor, Distribution of delta-5-3beta-hydroxysteroiddehydrogenase activity in the Graafian follicle of the sheep. Journal ofreproduction and fertility 43, 313 (May, 1975); N. B. Goldring et al.,Cholesterol side-chain cleavage P450 messenger ribonucleic acid:evidence for hormonal regulation in rat ovarian follicles andconstitutive expression in corpora lutea. Endocrinology 120, 1942 (May,1987); G. F. Erickson, A. J. Hsueh, Stimulation of aromatase activity byfollicle stimulating hormone in rat granulosa cells in vivo and invitro. Endocrinology 102, 1275 (April, 1978); herein incorporated byreference in their entireties). The measurement of gonadotropin receptorexpression provided a final analytical benchmark of the changesassociated with luteal transitions in vitro—the FSH receptor was acutelydownregulated by FSH while the LH receptor was unregulated andmaintained for 5 days post-hCG (J. S. Richards et al., Ovarianfollicular development in the rat: hormone receptor regulation byestradiol, follicle stimulating hormone and luteinizing hormone.Endocrinology 99, 1562 (December, 1976); herein incorporated byreference in its entirety). Given the paucity of human tissue, applyingmouse tissue to this engineered environment provides an alternativeapproach to study the molecular regulation of these important hormonetransitions.

Taken together, these results demonstrate that eIVFG is a powerfultechnique that allows the controlled study of human follicular andluteal endocrinology in ways that have not been possible to date. Thesestudies provide the first opportunity to track in vitro the hormone andtranscriptional changes that correlate with healthy follicles, using amodel that will provide new insights into mechanisms of fertility andreproductive function at the level of the single follicle. In additionto being able to control the power of the menstrual hormones in a dish,we were able to determine hormone production on a follicle by folliclelevel—including AMH which does not change during the menstrual cyclebecause of the aggregate production of this hormone by constantlyrenewing follicles. The mouse follicle cycle was extended to a 28 daysperiod, during which the hormone secretion pattern mimicked the humanmenstrual cycle. One hormone that differs between mouse and human isinhibin A, and this hormone and its regulating transcription factors,followed the mouse pattern precisely.

Experiments were conducted during development of embodiments of thepresent invention that succeeded in culturing human and mouse folliclesin a culture system that permits follicular phase growth, ovulation andluteinization. Using this approach iatrogenic effects ofchemotherapeutics and endocrine disruptors on follicles and oocyte canbe studied, mechanisms governing follicle selection can be explored, andan in vitro setting where oocyte maturation is supported leading tocompetent fertilizable eggs is created.

Acquisition of Human Ovarian Tissue

Human ovarian tissue was obtained following informed consent underInstitutional Review Board-approved protocols at NorthwesternUniversity. After surgical removal, ovaries were transported to thelaboratory in 48 hours at 4° C. in SAGE OFC holding Media (CooperSurgical, Trumbull, Conn.). In all cases, the ovaries were processedusing a standard technique in ovarian tissue cryopreservation in whichthe ovarian cortex is separated from the medulla(oncofertility.northwestern.edu/media/dissection-human-ovary-preparation-cryopreservation).

Human Follicle Culture and In Vitro Luteinization

Human follicles were isolated using a modified method as describedpreviously (R. Abir et al., Pilot study of isolated early humanfollicles cultured in collagen gels for 24 hours. Human reproduction 14,1299 (May, 1999); herein incorporated by reference in its entirety).Ovarian cortical strips were cut into 1 mm³ pieces in alpha MEM Glutamax(Invitrogen, Carlsbad, Calif.) supplemented with 1% Pen-Strep, 1% SerumProtein Substitute (SPS, Cooper Surgical, Trumbull, Conn.) and 1.5 IU/mlhCG. The tissue was then enzymatically digested by supplementing αMEMGlutamax with 1% Liberase TM (Roche, Indianapolis, Ind.) and 0.1% DNase(Worthington, Lakewood, N.J.) for 30 minutes at 37° C. After rinsing thecortex three times with fresh Sage holding medium, follicles were thenmechanically isolated from the cortex using insulin gauge needles, andencapsulated in 0.3% alginate (NovaMatrix, Philadelphia, Pa.).Encapsulated human follicles were transferred to a 96-well platecontaining 100 μl of growth media [αMEM Glutamax (Invitrogen, Carlsbad,Calif.) supplemented with 0.5 mg/ml fetuin, 0.3% human serum albumin(Cooper Surgical, Trumbull, Conn.), 5 μg/ml insulin, 5 μg/ml transferrinand 5 ng/ml selenium (Sigma-Aldrich, St. Louis, Mo.), and 5 mIUrecombinant FSH (A. F. Parlow, National Hormone and Pituitary Program,National Institute of Diabetes and Digestive and Kidney Diseases, USA)],and placed in an incubator at 37° C. and 5% CO2. Half of the culturemedium was exchanged every other day. The follicular phase ofdevelopment was tracked for each individual follicle by measuringestradiol, and once these levels plateaued, luteal conversion wastriggered by hCG administration for 36 hours and cultured for anadditional 15 days. After 36 hours incubation in maturation media, halfof the media was exchanged for growth medium without FSH.

Animals and Follicle Isolation

Animal experiments were performed using CD-1 female mice (HarlanLaboratories, Indianapolis, Ind.) that were housed in a controlledbarrier facility at Northwestern University's Center of ComparativeMedicine with constant temperature, humidity and light (12 h light/12 hdarkness) and food and water were provided ad libitum. All animalexperiments were approved by the Institutional Animal Care and UseCommittee and were in accordance with National Institutes of HealthGuidelines as well. Follicles (90 μm-120 μm) were mechanically isolatedfrom ovaries of 12- to 13-day old mice in L15 media (Invitrogen)supplemented with 1% fetal bovine serum (FBS, Invitrogen) and 10%streptomycin (Invitrogen), and encapsulated in cohorts of 10 folliclesin 0.5% w/v alginate as previously described (32). Briefly, individualfollicles were maintained in minimal essential medium (αMEM Glutamax,Invitrogen) supplemented with 1% FBS and 10% streptomycin(αMEM/FBS/Streptomycin) after isolation for at least 30 minutes beforeencapsulation. Follicles were then encapsulated in cohorts of ten (×10)in 0.5% (w/v) alginate (NovaMatrix, Philadelphia, Pa.). Follicles wererinsed with alginate and transferred to a 5 μl alginate drop made on apolypropylene mesh (McMaster-Carr, Atlanta, Ga.). The mesh wasimmediately lifted upside down over the calcium solution (50 mM CaCl2and 140 mM NaCl) for 2 min to allow for cross-linking to occur. Thealginate beads were removed from mesh and placed inαMEM/FBS/Streptomycin in the incubator to recover for at least 30minutes before transferred to culture medium.

Mice Follicle Culture and In Vitro Luteinization

Alginate encapsulated follicles were placed in individual wells of96-well plate containing 100 μl of growth medium [GM: αMEM Glutamaxsupplemented with 3 mg/ml BSA (MP Biomedicals, Solon, Ohio), 10 mIU/mlrecombinant FSH (A. F. Parlow, National Hormone and Pituitary Program,National Institute of Diabetes and Digestive and Kidney Diseases, USA),1 mg/ml bovine fetuin, 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/mlselenium (Sigma-Aldrich)] in a humidified atmosphere of 5% CO2 in air at37° C. for up to 14 days (in vitro follicular phase) upon which 1.5 IUhCG was administered to induce ovulation followed by an additional 15days of culture (in vitro luteal phase). Half of the culture media wasexchanged with fresh growth medium (GM) every other day and conditionedmedium stored at −20° C. Follicles were imaged at each medium changetime point using a Leica DM IRB inverted microscope equipped with 4× and20× objectives to assess growth and survival. Follicles that containedan oocyte surrounded completely by somatic cells would be defined aslive follicles. Dead follicles were defined by unhealthy oocytes, darkgranulosa cells, or lost structural integrity.

Immunohistochemistry

Follicles were fixed in Modified Davidson's Fixative (ElectronMicroscopy Sciences, Hatfield, Pa.) at the end of human follicle culturefor overnight at 4° C. And then follicles were dehydrated in increasingconcentrations of ethanol (50-70%) and embedded in paraffin. Thefollicles were sectioned at 5 μm serially and stained with hematoxylinand eosin. The cytoplasm to nucleus ratio and number of nucleus per 1cm² were quantified by Image J (National Institute of Health, Bethesda,Md.).

Hormone Assays

β-estradiol, progesterone, inhibin A, inhibin B, activin A and AMH weremeasured in spent medium by commercially available ELISA kits [estradioland progesterone (Calbiotech, Spring Valley, Calif.); AMH and inhibin B(Diagnostic Systems Laboratories); inhibin A (Beckman Coulter, Pasadena,Calif.); Activin A (Ansh Labs, Webster, Tex.)]. The limits ofsensitivity for estradiol, progesterone, inhibin A, inhibin B, Activin Aand AMH were 3.94 pg/ml, 0.22 ng/ml, <5.0 pg/ml, 7 pg/ml, 0.065 ng/ml,0.006 ng/ml, respectively. For each murine follicle culture, the beadswith at least 8 survived follicles on day 8 were selected. The mediawere then pooled together from different wells to do the hormone assays.However, the cultured media of five human follicles were measuredindependently.

RNA Isolation and Real-Time PCR

RNA was purified from follicles using the Qiagen RNeasy Micro Kitaccording to the manufacturer's protocol (Qiagen, Valencia, Calif.). RNAquality and quantity were assessed both by NanoDrop (Thermo Scientific,Wilmington, Del.) and BioAnalyzer 210 Expert (Agilent Technologies,Santa Clara, Calif.). From each condition, 100 ng of RNA was used forsynthesis of full-length cDNA using the AccuScript High Fidelity 1ststrand cDNA synthesis Kit (Aligent, Santa Clara, Calif.). Real-time PCRwas performed on the ABI PRISM® 7900HT Fast Real-Time PCR System(Applied Biosystems, Foster City, Calif.) using both the Taqman®Universal PCR Master Mix (Roche SOURCE, Indianapolis, Ind.) and Taqman®Gene Expression Assays (Applied Biosystems, Foster City, Calif.)according to the manufacturer's specifications. Gene-specific probeswere labeled with the FAM reporter dye at the 5′ end whereas anon-fluorescent quencher was linked to the 3′ end of the probe. Eachsample was performed in technical and biological duplicate and allresults were normalized to the endogenous control Rpl18. Analysis ofrelative gene expression was done using the comparative Ct (2-ΔΔCT)method (K. J. Livak, T. D. Schmittgen, Analysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta DeltaC(T)) Method. Methods 25, 402 (December, 2001); herein incorporated byreference in its entirety).

Statistics

Plotting of results and statistical analysis was done using GraphPadPrism (La Jolla, Calif.). All experiments were independently performedat least 3 times, unless otherwise noted. For comparisons betweengroups, a one-way ANOVA followed by Bonferroni post hoc test or unpairedStudent t test was performed. A P value of <0.05 was consideredstatistically significant.

Example 2 A Mammalian In Vitro Follicle Growth (IVFG) Assay PredictsAdverse Female Reproductive Outcomes Following Corexit EC 9500a Exposure

Drugs and Chemicals

Cisplatin (CDDP) and cyclophosphamide (CTX) were obtained fromSigma-Aldrich (St. Louis, Mo.) and used at final concentrations of: 0.2,1, and 5 μM. Nalbuphine and Raclopride were obtained from the NIHClinical Collection (South San Francisco, Calif.) and used at finalconcentrations of: 0.2, 1, and 5 μM. Corexit EC 9500A was obtained via aMaterial Transfer Agreement from Nalco (Sugar Land, Texas). CE was usedat final concentrations of: 5 ppm, 10 ppm, 25 ppm, 50 ppm, 75 ppm, 100ppm, 125 ppm, 150 ppm, 200 ppm, 250 ppm, 300 ppm, 350 ppm and 400 ppm,which correspond to 4.75 mg/L, 9.5 mg/L, 23.75 mg/L, 47.5 mg/L, 71.25mg/L, 95 mg/L, 118.75 mg/L, 142.5 mg/L, 180 mg/L, 237.5 mg/L, 285 mg/L,332.5 mg/L and 380 mg/L.

Animals and Follicle Isolation

All experiments were performed using CD-1 female mice (HarlanLaboratories, Indianapolis, Ind.) that were housed in a controlledbarrier facility at Northwestern University's Center of ComparativeMedicine under constant temperature, humidity and light (12 h light/12 hdark). Food and water were provided ad libitum. All animal experimentswere approved by the Institutional Animal Care and Use Committee andwere in accordance with National Institutes of Health Guidelines. Forfollicle isolation, ovaries were dissected from 14-15-day-old mice andcut into 4 pieces. The tissue was incubated for 1 hour in L15 media(Invitrogen, Carlsbad, Calif., USA) containing 2% Liberase TM (Roche,Indianapolis, Ind.) and 1% DNase I (Worthington, Lakewood, N.J.). Theenzymatic digestion was quenched by rinsing follicles in αMEM Glutamax(Invitrogen) containing 1% Fetal Bovine Serum (FBS, Invitrogen,Carlsbad, Calif.) and 0.5% Pen-Strep (Mediatech, Herndon, Va.). Type 5bmultilayer secondary follicles (140-175 μm in diameter) were manuallycollected and cultured individually in wells of a 96-well plate(Pedersen and Peters, 1968; herein incorporated by reference in itsentirety). Follicles were grown in 100 μl of growth media (GM) comprisedof αMEM Glutamax supplemented with 10 mIU/ml recombinantfollicle-stimulating hormone (rFSH; A. F. Parlow, National Hormone andPituitary Program, National institute of Diabetes and Digestive andKidney Diseases), 1% Pen-Strep, 5 μg/ml insulin, 5 μg/ml transferrin,and 5 ng/ml selenium (Sigma-Aldrich, St. Louis, Mo.).

Follicle Culture, Growth and Survival

Follicles were cultured at 37° C. in a humidified atmosphere of 5% CO₂in air for up to 6 days. All chemical exposures were initiated on day 2of culture. Half of the culture media was replaced with fresh GM everyother day and spent media was stored at −20° C. for use in steroidhormone assays. Follicles were imaged at each media change using a LeicaDM IRB inverted microscope equipped with 4× and 20× objectives (LeicaMicrosystems, Heidelberg, Germany) to assess survival, morphology andgrowth. Live follicles were defined as those that contained an oocytesurrounded completely by somatic cells. Dead follicles wherecharacterized by dark granulosa cells, granulosa cells that failed toproliferate, or an oocyte that had dissociated completely from thefollicle structure (Lenie et al. 2008′ herein incorporated by referencein its entirety). The percent of surviving follicles was calculated asthe number of live follicles out of the total number of folliclesplated. Antral cavity formation was assessed morphologically andcharacterized by the differentiation of outer layers of mural granulosacells and inner layers of cumulus granulosa cells surrounding the oocyteas well as the presence of a fluid-filled structure.

Hormone Assays

To assay hormones secreted by cultured follicles, spent culture mediafrom viable follicles were pooled both on day 4 and day 6. β-estradiol(E2) was measured using a commercially available Estradiol (E2) ELISAKit (Calbiotech, Inc. Spring Valley, Calif.) according to manufacturer'sprotocol. All assays were run in duplicate. The assay sensitivity is3.94 pg/ml, and medium collected from wells without follicles was usedas a negative control.

In Vitro Oocyte Maturation

In vitro maturation (IVM) was performed after 6 days of culture.Briefly, half of the medium was replaced with IVM medium (αMEM Glutamaxcontaining 5 ng/ml epidermal growth factor, 1.5 IU/ml human chorionicgonadotropin (HCG) and 5% FBS) and follicles were incubated at 37° C. ina humidified atmosphere of 5% CO₂ in air for 14-16 h. Oocytes were thenremoved from the cumulus cells by treatment with 0.3% hyaluronidase. Themeiotic stage of each oocyte was scored morphologically using the LeicaDM IL microscope. Oocytes that remained arrested in prophase of meiosisI as evidenced by an intact germinal vesicle (GV) were classified asGV-intact oocytes. Oocytes that resumed meiosis and reached metaphase ofmeiosis II (MII) as evidenced by polar body extrusion were classified asMII-arrested eggs. Those oocytes that resumed meiosis but did not reachMII, as evidenced by lack of a both a GV and a polar body, were referredto as oocytes that had undergone GV breakdown (GVBD). Degenerate oocyteswere also documented. The meiotic progression distribution is reportedas the number of oocytes in each stage over the total number offollicles that were in vitro matured.

Egg Cytoskeleton Analysis

To examine the microtubule and actin cytoskeleton, cells were processedas described previously (Hornick and Duncan, 2012; herein incorporatedby reference in its entirety). Briefly, oocytes were fixed in 3.8%paraformaldehyde (Electron Microscopy Sciences, Hatfield, Pa., USA)containing 0.1% Triton X-100 (Sigma-Aldrich) in 1× phosphate-bufferedsaline (PBS) for 1 hr at room temperature (RT). After fixation, oocyteswere washed with blocking buffer (BB) containing 0.3% BSA, 0.02% NaN₃,and 0.01% Tween-20 in 1×PBS and then incubated in permeabilizationsolution containing 0.3% BSA, 0.1% Triton X-100 and 0.02% NaN₃ for 15minutes. After rinsing with BB, oocytes were incubated in a 1:100dilution of Alexa Fluor 488-conjugated α-tubulin Rabbit mAb for 1 h atRT to detect the microtubule cytoskeleton (Cell Signaling, Boston,Mass.). F-actin was detected by simultaneous incubation in a 1:50dilution of rhodamine phalloidin (Invitrogen). All antibodies werediluted in BB. Oocytes were washed in BB after antibody incubations andmounted in Vectashield containing DAPI to detect DNA (VectorLaboratories, Burlingame, Calif., USA). Images were acquired with aLeica SP5 inverted laser-scanning confocal microscope equipped with a40× objective and Confocal LAS AF software (Leica Microsystems,Heidelberg, Germany). Additional image processing was done using Image Jsoftware (National Institutes of Health, Bethesda, Md.). Spindlemorphology was scored and normal spindles were defined as those thatwere bipolar and had chromosomes tightly aligned on the metaphase plate.

Ovarian Stromal Cell Isolation and Survival

Ovarian stromal cell isolation was carried out as described previouslywith minor modifications as detailed (Tingen et al. 2011; hereinincorporated by reference in its entirety). Ovaries harvested from day21-22 mice were placed in L15 media containing 0.5% Pen-Strep and 1%FBS. Granulosa cells were released by mechanical disruption of theovaries using two insulin gauge needles. The remaining tissue wasdissociated in 1× Trypsin/EDTA-Hanks (Invitrogen) containing 1% LiberaseTM (source) and 1% DNase I (source) with gently rocking for 1 hr at 37°C. in a humidified atmosphere of 5% CO₂ in air. The enzymatic reactionwas stopped by addition of 10% FBS and the solution was passedsequentially through 70 μm and 20 μm filters (Gorlitz, Germany). Theflow through was then pelleted by centrifugation for 5 min at 1500 rpmand rinsed with the following growth media: DMEM/F12 supplemented with1M HEPES, 20 mg/ml androstenedione, 4 mg/ml hydrocortisone, 6.5 μg/mlinsulin, 6.5 μg/ml transferrin, and 6.5 ng/ml selenium (Sigma-Aldrich,St. Louis, Mo.). The cells were pelleted again and rinsed with growthmedia containing 10% heat-inactivated FBS. Cells were then resuspendedin 1-2 ml of growth media with FBS and plated in 96-well polystyreneplates at an approximate density of 50,000 cells per well. Cells wereincubated overnight at 37° C. in a humidified atmosphere of 5% CO₂ inair. The following day the media was replaced with growth mediacontaining different concentrations of CE. Growth media was changedevery other day, and the MTS CellTiter 96 Aqueous Non-Radioactive OneSolution Assay (Promega Corp., Madison, Wis.) was used to evaluate cellviability on day 4 of culture according to the manufacturer'sinstructions.

HeLa Cell Culture and Survival

Human cervical cancer cells (HeLa cell line; ATCC, Manassas, Va.) werethawed and plated in 96-well plates at an approximate density of 50,000cells per well. CE exposure and the cell viability assay were performedexactly as described for stromal cells. The only modification was thatthe cell viability assay was performed on Day 2 for HeLa cells sincethey proliferate more rapidly than primary cell lines.

Statistical Analysis

Plotting of results and statistical analysis was done using Graph Prism(La Jolla, Calif., USA). Antral cavity formation, estradiol production,and survival following CDDP and CTX exposure were analyzed using atwo-way analysis of variance (ANOVA) followed by a Bonferroini posttestfor single time point comparisons. HeLa cell, stromal cell, and folliclesurvival were determined by sigmoidal dose-response (variable slope)curve followed by an F-test. Meiotic progression and spindle morphologywere analyzed using a one-way analysis of variance (ANOVA). In allcases, a p-value of less than 0.05 was considered statisticallysignificant.

IVFG can be Used to Identify Chemicals that have Potential AdverseReproductive Outcomes

To confirm the utility of the IVFG system, we validated the assay bydetermining the effects of two common chemotherapeutics,cyclophosphamide (CTX) and cisplatin (CDDP), that are known to be toxicto the ovary and to follicles specifically (Kerr et al. 2012, Hartmannand Lipp 2003; herein incorporated by reference in their entireties).Isolated stage 5b mouse follicles were grown in the presence of 0.2, 1,5 μM CTX and CDP, and survival rate was quantified based on folliclemorphology (FIG. 8). Follicle survival decreased in a dose-dependentmanner during culture in CTX and CDDP. Exposure to 1 μM and 5 μM CTXresulted in a significant decrease in survival, from 95.40% to 57.23%and 36.41%, respectively, compared to control follicles by day 4 (FIG.8A; P<0.001). By day 6, this decrease in survival was even morepronounced with only 32.43% and 11.49% follicles surviving in the 1 μMand 5 μM CTX treatment groups, respectively. At this time, thedifference in survival between follicles cultured in 0.2 μM CTX comparedto control was also statistically significant (FIG. 8A; 71.8% and95.40%, respectively, P<0.05). A similar trend in decreased folliclesurvival after 4 and 6 days of culture was observed following CDDPtreatment compared to control follicles (FIG. 8B). This survivalreduction due to CDDP exposure, however, was only significant at the 1μM and 5 μM doses (FIG. 8B; 76.31 and 47.83% on day 4 and 58.38% and12.56% on day 6, respectively, P<0.05 compared to control). The dataimply that increasing concentrations of both CTX and CDDP adverselyimpacted follicle survival.

In addition to survival, CTX and CDP exposure also affected folliclemorphology. In control follicles grown without CDDP or CTX, granulosacells proliferated and differentiated into mural and cumulus cellsconcomitant with antral cavity formation (FIGS. 2C, D). In contrast,increasing concentrations of CTX and CDDP resulted in disorganizedfollicles with dark granulosa cells and little evidence of antral cavityformation (FIGS. 2C, D). The most severe phenotype, observed first at 1μM CTX and 5 μM CDDP, was the complete release of the oocyte from thefollicle (data not shown and FIG. 8D).

To confirm that the follicle morphology and survival results obtainedfollowing CTX and CDDP exposure were specific and not simply due to ageneral toxicity response, follicles behavior was examined, when exposedto two FDA-approved pharmaceuticals, Nalbuphine and Raclopride that,unlike CTX and CDDP, have no known documented reproductive toxicity(Borovskaya et al. 2004; Kenney et al. 2001; Kociba and Sleight 1971;Meirow et al. 1999; Meirow and Nugent 2001; Miller 1980; Sklar 2005;Wallace et al. 1989; Yanulevich 1983; Yeh et al. 2006; Yucebilgin et al.2004; herein incorporated by reference in their entireties). Stage 5bmouse follicles cultured in the presence of 0.2, 1, 5 μM Nabulphine andRaclopride, in contrast to those grown in the presence of CTX and CDDP,were similar to controls in terms of survival and morphology (FIG. 9;P>0.05). Thus, this IVFG system can distinguish differences in howspecific chemical exposures influence follicle health and survival.

Dose-Dependent Corexit Exposure Impacts IVFG Reproductive Endpoints

To determine the effect of CE on follicle growth and function, follicleswere exposed to increasing concentrations of CE, ranging from 0 ppm to400 ppm. Overall follicle survival decreased significantly in adose-dependent manner (FIG. 10A). By day 4 of culture, for example,survival decreased from 99.08% in control follicles to 50.76%, 11.30%and 0% in follicles cultured in 75 ppm, 100 ppm and 125 ppm CE,respectively (FIG. 10A; P<0.001). By day 6, this decrease in survivalwas even more pronounced with 37.10% and 0% follicles surviving in the75 ppm and 100 ppm CE treatment groups, respectively. At this time, thedifference in survival between follicles cultured in 25 and 50 ppmcompared to control were also statistically significant (FIG. 10A;76.64%, P<0.05 and 61.83%, P<0.001, respectively). Based on these datait was determined that the concentration at which half of the folliclessurvived at day 6, or the median lethal dose (LD₅₀) with 95%confidential interval, was 57.37±8.94 ppm (FIG. 10B, triangles). Giventhis LD₅₀, subsequent experiments were done with CE concentrations of≦75 ppm.

In addition to affecting follicle survival, exposure to increasingconcentrations of CE altered follicle morphology and cellulardifferentiation (FIG. 10C). Follicles grown in 10 ppm CE appearedmorphologically normal and indistinguishable from controls,demonstrating appreciable granulosa cell proliferation and antral cavityformation (data not shown). Follicles exposed to 25 ppm CE also appearedhealthy, but their antral cavities appeared smaller compared to those incontrol follicles (FIG. 10C). In contrast, exposure to 50 ppm and 75 ppmCE resulted in severe follicle disorganization (FIG. 10C).

To better understand how CE affected follicle growth and morphology, thepercent of follicles that formed antral cavities when exposed to thedispersant was quantified (FIG. 11A). Antral cavity formation wasassessed between day 4 and 6 of culture, as this is when many folliclesform this characteristic structure (FIGS. 2B, 2D, 4C). On day 4 ofculture, only follicles grown in 75 ppm CE showed a significantreduction in antral cavity formation compared to control (FIG. 11A,10.15% vs. 41.18%, respectively; P<0.05). By day 6, however, significantreductions in antral cavity formation were also observed at lowerconcentrations of CE. For example, even at 25 ppm CE, the percentage offollicles that formed an antral cavity was 44.4% compared to controlfollicles in which it was 77.6% (FIG. 11A, P<0.001). In controlfollicles, a significant increase in the percent of follicles that formantral cavities occurs between day 4 and day 6 of culture (FIG. 11A,41.18% to 70.84%, respectively; P<0.05). However, when antral cavityformation was compared between day 4 and day 6 for each CEconcentration, it was found that this increase did not occur whenfollicles were exposed to CE concentrations greater than 25 ppm (FIG.11A). Taken together these data, therefore, indicate that CE exposurehas a dose-dependent effect on follicle morphology, with a specificconsequence on cellular differentiation and antral cavity formation.

Corexit Exposure Affects Follicle Hormone Production

A major hallmark of follicle function is the ability of the granulosacells to synthesize steroid hormones (Zeleznik 2004; herein incorporatedby reference in its entirety). Therefore to determine how CE exposureaffected follicle function, we measured estradiol production in thespent culture media as estrogen is secreted by growing follicles and itsconcentration increases with follicle development and differentiation(Cortvrindt and Smitz 2002; herein incorporated by reference in itsentirety). Estradiol levels were low on day 4 but increased dramaticallyby day 6, coincident with the large increase in follicle growth anddifferentiation that is also observed during this time (FIGS. 4C, 5). CEexposure on day 4 did not have a significant effect on baselineestradiol levels. However, at day 6 of culture, CE exposures of 10 ppmand 25 ppm resulted in a slight but significant reduction in estradiolproduction compared to control (3127 pg/ml and 3097 pg/ml compared to4007 pg/ml, respectively, p<0.05; FIG. 11B). This negative impact onestradiol production was even more dramatic at CE concentrations of 50ppm and greater where estradiol production was only between 842.9 pg/mland 1094 pg/ml, p<0.001; FIG. 11B). In fact the large relative averageincrease in estradiol production, which typically occurs between day 4and day 6, was abrogated significantly in follicles cultured in ≧50 ppmCE (p<0.05; FIG. 11B). These results indicate that similar to follicularmorphology and development, CE exposure influences hormonal homeostasis.

Corexit Exposure Causes Meiotic Defects in the Oocyte

In addition to producing hormones that support follicle development andendocrine feedback, a prime function of the ovarian follicle is toproduce a healthy female gamete. One hallmark of a fully-grown oocyte isits ability to resume meiosis and reach MII in response to gonadotropinstimulation. It was examined how CE exposure during IVFG impacted thisprocess, also referred to as meiotic maturation. To do this, meioticresumption was stimulated in follicles that had been exposed todifferent doses of CE on day 6 and then scored meiotic progression. Thecategories that were scored GV, GVBD/MI, and MII which correspond to thefollowing meiotic stages: prophase I-arrested, prometaphase I ormetaphase I, and metaphase II, respectively (FIG. 12A). The number ofcells that were degenerated was also scored. Only those cells that havecompleted the first meiotic division with emission of the first polarbody and reached MII have the ability to be fertilized (Ducibella andBuetow 1994; herein incorporated by reference in its entirety). Incontrol follicles that were not exposed to CE, 78%±4.243% of the gametesreached MII (FIG. 12B). This percentage was unchanged in follicles thatwere exposed to 10 ppm and 25 ppm of CE. However, at 50 ppm and 75 ppmonly 21.80% and 13.90% of the oocytes reached MII, respectively(p<0.001; FIG. 12B). This decrease in mature gametes was insteadaccompanied by a significant increase in the percent of degeneratedcells (FIG. 12B). CE exposure did not affect the percentage of cellsthat were in the GV or GVBD/MI stage (FIG. 12B). These data imply thatat higher concentrations, CE negatively impacts the ability of thefollicle to produce a viable and mature egg.

Although CE doses of 50 ppm and 75 ppm significantly compromised meioticmaturation, there were still some MII eggs produced. To betterunderstand the quality of the resulting eggs, a more detailed analysisof their cytoskeleton morphology was performed (FIGS. 6C, D).Specifically the structure of the meiotic spindle and cortical actinfollowing exposure to increasing CE concentrations was examined (FIG.12C). A healthy egg has a symmetric and bipolar spindle with chromosomestightly aligned on the metaphase plate (Lenie et al. 2008; hereinincorporated by reference in its entirety). Such a structure isessential for mediating proper chromosome segregation upon fertilizationto produce a haploid female gamete. In addition, the actin cytoskeletonis cortical and polarized in the region overlying the meiotic spindleand chromosomes. No differences in spindle structures in MII eggs fromfollicles exposed to 10 ppm and 25 ppm CE were observed compared tocontrols. However, abnormal spindles were observed following exposure tohigher concentrations of CE (FIGS. 6C, D). In follicles grown in 75 ppmCE, no normal spindle morphology was observed in the resulting eggs.Instead spindles in these eggs lacked an organized bipolar structure andhad significant chromosome misalignment (FIG. 12C). In contrast to themeiotic spindle which appeared to be highly sensitive to CE, corticalF-actin was not (FIG. 12C).

The Ovarian Follicle is More Sensitive to Corexit than Cell Lines

In toxicity studies, cell lines are often used for in vitro drugscreening and toxicity studies (Allen, et al 2005; herein incorporatedby reference in its entirety). Primary ovarian stromal cells and HeLacells were grown in CE concentrations ranging from 5 ppm to 400 ppm andquantified their survival using a cell viability assay. For both primaryovarian stromal cells and HeLa cells, the data was fit to a variablesigmoidal dose-response curve and an F-test was performed to determinesignificance (p<0.0001; FIG. 12B). It was calculated that the LD₅₀ with95% confidential interval for primary ovarian stromal cells was151.9±32.25 ppm and that for HeLa cells was 194.1±22.8 ppm. Thisobserved difference in survival between HeLa cells and primary ovarianstromal cells was statistically significant (p<0.05). Moreover, the LD₅₀of follicles was significantly lower than either of the somatic cellcounterparts examined (p<0.0001; FIG. 12B). These data highlightdistinct differences in how cells behave in response to CE and suggestthat transformed cells are more resistant than primary cells. Moreover,the ovarian follicle—which contains both somatic and germ cellcomponents—is particularly sensitive to CE.

Example 3 Human Fallopian Epithelium Response In Vitro to Mimetic of theHuman Menstrual Cycle

The human fallopian tube (named oviduct in non-primate species) is acritical organ in the female reproductive tract that facilitates thetransport of the cumulus-oocyte complex (COC), sperm, and embryos(Hunter, 2005; herein incorporated by reference in its entirety). Theoviduct consists of distinct segments that contribute to the process ofgamete transport and fertilization including the fimbriated ends thatlie adjacent to the ovary, the ampulla, the ampulla-isthmus junction,and the isthmus portion (Croxatto, 2002; herein incorporated byreference in its entirety). The fimbriae wrap around the ovary andtransport the COC into the ampulla portion (Croxatto, 2002; hereinincorporated by reference in its entirety). The isthmus primarilyparticipates in the binding and release of spermatozoa through a seriesof specific carbohydrate residues that generate a sperm reservoir(Talbott et al., 2003; herein incorporated by reference in itsentirety). The embryo also transiently attaches to the oviductalepithelium and is transported to the uterus for implantation (Velasquezet al. 2001; herein incorporated by reference in its entirety).Therefore, understanding oviductal biology using proper in-vitro modelsis crucial to improving reproductive health.

The entire epithelium is regulated by hormones as well as by the gametesand the embryos that interact with the epithelium (Abe et al, 1999;Bauersachs, 2004). Estrogen and progesterone bind to their receptors, ERand PR, in the epithelium to induce alterations in gene function suchas, oviductal glycoprotein 1 (OVGP1), IGF1, and ciliary beating(Bylander, 2010; Mahmood, 2008; Nakahari, 2011; herein incorporated byreference in their entireties). OVGP1, which is secreted in response toestrogen, positively influences the capacitation of sperm and itsmotility during fertilization (Erickson-Lawrence, et al.; 1989; Verhageet al., 1997; Verhage et al., 1990; herein incorporated by reference intheir entireties). Experiments were conducted during development ofembodiments of the present invention to demonstrate functionalbiological stimulation of primary human fallopian tube cultures to themenstrual cycle. The culture system for the first time integrates thebiology of the maturing ovarian follicle with the downstream biologicalfunction of the fallopian tube in terms of cilia action, geneexpression, and secreted factors.

Human fallopian epithelia layers, which included epithelia cells andstromal cells, were cultured for 28 days on TRANSWELL membrane inserts(FIG. 13A). The epithelium structure was clearly identifiable andmaintained strong ERα, PR, and OVGP1 expression after culture for 28days (FIG. 13B). In order to study functional changes in the humanfallopian epithelia tissue in response to human sex steroids, themenstrual cycle was divided into four seven day blocks as follows: lowE2 (0.1 nM) and high E2 (1 nM) to mimic the follicular phase; E2 1 nM+P410 nM and E2 0.1 nM+P4 50 nM to mimic the luteal phase (Baerwald, 2012;herein incorporated by reference in its entirety). Using the humanfallopian epithelial culture system cilia beating frequency, OVGP1expression and IGF1 secretion were stimulated by E2 and suppressed by P4(FIG. 14) (Mahmood et al., 1998; Verhage et al., 1990; Shao et al.,2007; herein incorporated by reference in their entireties). Ciliabeating was maintained after 7 days in culture. Cilia beating frequency(CBF) significantly increased with E2 treatment, and decreased upon theaddition of P4 (FIG. 14A, B). OVGP1 expression in the fallopian lysatealso demonstrated induction from E2 treatment, and was suppressed by theaddition of P4 (FIG. 14C). Similar to OVGP1 expression and CBF, IGF1secretion was induced by 1 nM E2 and attenuated by the addition of P4(FIG. 14D). These data indicate that human fallopian epithelium culturedon microporous membrane inserts can be used to model biologicalresponses to hormonal stimulation.

The previous section demonstrated that human fallopian tube grown onmicroporous membrane inserts functionally responded to combinations ofestrogen and progesterone over 7 days. Next, a stepwise increase insteroid hormones was designed to mimic the human menstrual cycle over 28days (FIG. 15A). Similar to individual 7 days cultures, the prolongedculture, exposed to the menstrual cycle in these four stages,demonstrated that OVGP1 (FIG. 15B) and IGF1 (FIG. 15C) were bothstimulated by E2 and suppressed by the addition of P4 in the presence ofE2.

Maturing ovarian follicles secrete hormones that dictate thefluctuations of the menstrual cycle. In vitro maturation of follicles inalginate hydrogels can support the growth and steroidogenic function ofthe in vivo follicle (Xu, et al., 2006A; Xu, et al., 2006B; Xu, et al.,2007; herein incorporated by reference in their entireties). Due to thefinite and limited access to human ovarian follicles for research, anengineered system of mouse in vitro follicle culture was utilized thatdemonstrated the ability to mimic the human follicular and luteal phasein terms of steroid hormone and peptide hormone. To test the response offallopian epithelium tissue insert cultures to physiologic hormonestimulation from ovarian follicles, a co-culture was established (FIG.16A). Although the cycle was shortened to 14 days, the E2 and P4 patternin the follicle culture system matched the human menstrual cycle pattern(FIG. 16B). E2 levels rose during the first seven days mimicking thefollicular cycle. In-vitro maturation (IVM) was stimulated using hCG onthe evening of day 7 promoting an increase in progesterone productionduring the luteal phase for another seven days. The morphology offallopian epithelial layers in the co-culture system demonstrated cellviability after 7 days and 14 days of culture (FIG. 16C). Human IGF1(hIGF1) secretion was used as a functional biomarker of hormoneresponsiveness for the fallopian tube cultures. hIGF1 expressioncontinually increased before IVM when estrogen levels were high and thendeclined after IVM indicating progesterone mediated repression (FIG.16D). Therefore, the human fallopian tube can be co-cultured with themouse ovarian follicle to better mimic hormone fluctuations thatnormally occur as part of the human menstrual cycle. In additionalembodiments, one could interrogate the follicle and fallopian 3Dco-culture system with additional female reproductive tissues in amicrofluidic culture system.

The fallopian tube is an important reproductive target forpharmaceutical intervention and access to reliable models of humantissue are essential for preclinical studies that might enhancefertility, provide contraception, reduce ectopic pregnancy, and impactserous cancer formation. The described system used normal humanfallopian tube tissue from women undergoing hysterectomy on microporousTRANSWELL inserts with maintenance of viability and hormone receptorexpression for up to 28 days in culture. Hormonal fluctuations of thehuman menstrual cycle were modeled using three different methods. First,estrogen and progesterone were used in four combinations for seven daysto mimic the different stages of menstrual cycle. Second, hormonecombinations were changed in a step-wise manner to mimic a 28 daysmenstrual cycle. Lastly, the fallopian tube epithelium tissue wereexposed to in vitro cultured mouse follicles engineered to secretehormones in a manner that phenocopies a shortened human menstrual cycle.Primary human fallopian epithelium maintained ciliated and secretoryphenotypes and functionally responded to multiple hormone stimulationprocedures through 28 days of culture.

Specifically, estrogen increased OVGP1 expression, IGF1 secretion, andciliary beating rates, while the addition of progesterone decreasedthese functional markers. In addition, co-culture of human fallopianepithelia with ovarian mouse follicles proved to be an effective modelof fallopian and follicle interactions.

Human fallopian tube was cultured with ovarian murine follicles to modela physiologic source of hormones. Since steroids estrogen andprogesterone are conserved across species, the mouse follicle co-culturewith the human fallopian epithelium in this study provides a novelapproach to not only monitor the response of human fallopian tube cellsto follicular fluid from bin vitro matured and ovulated follicles, butalso during growth of the follicle. Additional factors in conditionedmedium from in vitro matured follicles and follicular fluid, such asprostaglandins (Seibel et al., 1984; herein incorporated by reference inits entirety), can also be monitored in future analysis to determinetheir role on inflammation and cancer initiation (Levanon et al., 2010;King et al., 2011; herein incorporated by reference in theirentireties). These studies provide in vitro confirmation that culturedfallopian tube epithelium responds to the menstrual cycle hormones in asimilar manner to tissues collected from women at different stages ofthe menstrual cycle (Lyons et al., 2002; herein incorporated byreference in their entireties).

In vitro models of fallopian epithelium in the menstrual cycleenvironment provide an important tool for understanding women's health.The 3D model can be extended to study normal physiology, infertility,and cancer. Effects of environmental toxins, such as cigarette smoke,and pharmaceuticals, such as emergency contraception, on the fallopianepithelium can be studied both dependently and independently of ciliabeating (Leng et al., 1998; Knoll et al., 1998; herein incorporated byreference in their entireties). The role of oviductal secretions onsperm capacitation and transport could be uncovered in this model andprovide a system for studying new drugs that modulate sperm to enhanceor prevent fertilization. Early development of the embryo occurs in thefallopian tube and may be negatively impacted by environmentalpollutants or pharmaceutical drugs that can be studied safely in an invitro human model system. The secretory cells of the fimbria offallopian tubes are potential sites for serous cancer formation and therole of steroid hormones may influence cancer formation and/orprevention (Corner et al., 1998; Yamanouchi, 2010; herein incorporatedby reference in their entireties). The human fallopian tube culturesystem provides a robust model for studying the biology of thesereproductive health concerns. In certain embodiments, one couldintegrate the fallopian epithelium and follicle cultures with uterinetissues to establish an integrated microphysiologic female reproductivetract for study in combination with non-reproductive tissues providing awhole body model of hormone function.

Human Fallopian Tissue Culture

Human fallopian tissue was obtained from routine hysterectomies fromwomen in their reproductive years (ages range from 26 to 50 years old)and who had not undergone exogenous hormonal treatment for at leastthree months prior to surgery. Informed consent was obtained from eachpatient and approval granted by the ethics committee of the NorthwesternUniversity. Human fallopian tissue was kept in the DMEM/F12 medium with10% FBS on ice and processed within 24 hours. The tissue was washed withwarm PBS solution two times before being transferred into warmdissection medium (Liebowitz 15 with 0.5% P/S and 10% FBS). Aftercleaning connective tissue from outside of the fallopian tissue, thefallopian tube was cut open and the inner epithelia layer wasmechanically dissected using forceps. The epithelia layer wastransferred to phenol red free DMEM/F12 with 0.3% BSA, 1% P/S and ITS (5μm/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium) (Sigma-Aldrich,St. Louis, Mo.) medium and cut into 2×2 mm pieces for further culture.Tissue was cultured on 0.4 μm pore Millicell inserts (PICM03050, EMDMillipore Co, Billerica, Mass., USA) twelve-well plates with culturemedium in a 37° C. and 5% CO2 incubator. Estrogen (E2) and progesterone(P4) used in tissue culture experiments were purchased from Sigma (St.Louis, Mo.).

Histology

Cultured fallopian epithelia layers were fixed with Modified Davison'sfixative (Electron Microscopy Science Inc., Hatfield, Pa.) for 24 h at4° C. and then processed and embedded in paraffin. H&E staining wasperformed using standard methods. Immunofluorescence staining (IF) wasperformed and visualized as previously described (Kim et al., 2013;herein incorporated by reference in its entirety). The primaryantibodies used for the IF staining were as follows: ER alpha (Abgen,Pittsburgh Pa.) for 1:70 dilution, PR (Dako, Carpinteria, Calif.) for1:200 dilution, Ki67 (abcam, Cambridge, Mass.) 1:100 dilution and OVGP1(LsBio, Seattle, Wash.) 1:100 dilution. Tissue stained without addingprimary antibody was used as negative control.

Immunoblot Analysis

Cultured human fallopian epithelia layers were homogenized with aminipestle in ice-cold lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mMNaCl, 10% glycerol, 1% NP-40, 2 mM EDTA). Lysate normalized for proteincontent was loaded into NuPAGE 4-12% gradient Bis-Tris precast SDS-PAGEgels and transferred to a nitrocellulose membrane (Life Technologies,Carlsbad, Calif., USA). Blots were probed by polyclonal anti-OVGP1antibody (LsBio, Cambridge, Mass.) overnight in 4° C. followed byanti-rabbit secondary antibody conjugated to horseradish peroxidase(Zymed, San Francisco, Calif.). Proteins were detected by ECL primer (GEHealthCare Life Sciences, Pittsburgh, Pa.) and exposed to X-ray film(Kodak, Rochester, N.Y.). The same blot was stripped using strippingbuffer (Thermo Scientific, Hanover Park, Ill.) and re-probed withmonoclonal anti-α-tubulin (Sigma, St. Louis, Mo.) followed by ananti-mouse secondary antibody conjugated to horseradish peroxidase. NIHImageJ Imaging Software was used to quantify the density of western blotbands. Conditioned medium from fallopian epithelia cultures wascollected and probed via western blot analysis using a polyclonalanti-hIGF1 anti-body (abcam, Cambridge, Mass.). The blot was detected bythe same method described above.

Glucose ELISA

Fallopian epithelia layers cultured on TRANSWELL inserts werepre-cultured in the alpha-MEM with 0.3% BSA, ITS and 1% P/S medium fortwo days. Cell culture medium was collected for the glucose ELISA assay(abcam, Cambridge, Mass.). Medium was diluted 1:500 and ELISA wasperformed according to the protocol provided by the kit. Glucoseconsumption results were used to normalize the hIGF protein secretion incell culture media to account for slight size differences in culturedfallopian tube epithelial layers.

Fallopian Epithelia Cilia Beating Quantification

Fallopian epithelia layers were cultured in estradiol (E2) 1 nM, E2 1nM+progesterone (P4) 10 nM, E2 0.1 nM+P4 50 nM or with solvent onlynegative control for 7 days. Cilia beating movies were acquired using anAndor Spinning Disk Confocal with 5 ms exposure time and 5 ms readouttime. Each movie consists of 100 individual frames. Five movies indifferent areas of the sample were taken. The Multiple Kymograph pluginfor the NIH ImageJ software was used to quantify cilia beatingfrequency. In each movie, 8 clusters of cilia were picked for the ciliabeating quantification.

Follicle Co-Culture with Fallopian Epithelia Layer

CD-1 mice were purchased from Harlan Laboratories (Madison, Wis., USA).All procedures involving mice were approved by the NorthwesternUniversity Animal Care and Use Committee. Mice were housed and bred in abarrier facility within Northwestern University's Center of ComparativeMedicine (Chicago, Ill., USA) and were provided with food and water adlibitum. Temperature, humidity, and photoperiod (14 L:10 D) were keptconstant. Day 14 CD1 mice ovaries were removed and 150 um-180 um sizefollicles were isolated from the ovaries. 5 follicles were encapsulatedinto 0.5% (w/v) alginate beads for further culture. The alginate beadwas placed in one well of a 12 well plate and the fallopian tubeepithelial layer tissue was placed on the 0.4 um insert membrane. Theinsert containing the fallopian tube culture was placed into the samewell as the follicle in the alginate bead. Medium in the 12-wellco-culture was composed of follicle culture medium, made from alpha MEMwith 0.5 mg/ml fetuin (Sigma-Aldrich, St. Louis, Mo.), 0.3% BSA, ITS,and 10 mIU/ml recombinant human FSH supplement for 7 days. On day 7, themedium was changed to in vitro maturation medium, containing 1.5 IU HCGin follicle growth medium for 16 hours. After 16 hours, the medium waschanged back to follicle growth medium without rhFSH. The follicles, nowluteinized due to exposure to HCG, were continually cultured for another7 days. Every other day 50 μl of the medium was collected for the E2, P4ELISA test and western blot for hIGF1. The pictures for the follicleswere taken every other day.

Steroid Hormone ELISA

E2 and P4 in follicle culture medium were detected by the E2 and P4ELISA kit (Calbiotech, CA, USA) using the manufacturer's protocol.

Statistical Analyses

The cilia beating quantification and western blot band densitometryquantification were analyzed using One-way ANOVA followed by Tukey'smultiple comparisons test. A p-value less than 0.05 was consideredstatistically significant.

Example 4 Human Uterine Cultures in 3D Microphysiologic Setting

Experiments were conducted during development of embodiments of thepresent invention to establish static 3D cultures of uterine cells(e.g., myometrial cells, endometrial epithelial cells, and endometrialstromal cells) and to determine the biological response of the cells insuch a system to stimuli such as estradiol and progesterone.

Static 3D cultures of uterine cells are established in Transwellcultures with endometrial epithelial and endometrial stromal cells inthe upper chamber and myometrial cells in the lower chamber.

During a 28-day female reproductive cycle, hormone levels (e.g.,estrogen and progesterone) are in flux and the levels of varioussecreted factors varies (FIG. 17). Experiments were conducted using thestatic 3D cultures on polystyrene scaffolds described above. Morphologicand hormonal responses of cultured cells were assessed in response tofluctuating levels of estradiol (E2) or progesterone (P4).Morphologically, the 3D endometrial cultures were composed of a mixedpopulation of epithelial and stromal cells. 3D myometrial culturescomprised of myometrial cells (FIG. 17). Cells expressed vimentine andprogesterone receptor to in both endometrial and myometrial cells in 3D.A 45-plex Luminex assay identified numerous factors secreted andregulated by hormones (FIG. 18).

The procedure for establishment of 3D endometrial and myometrialcultures on decellularized matrix involved the isolation of epithelialand stromal cells from endometrial tissue and myometrial cells frommyometrial tissue followed by expansion of the cells in 2D, andsubsequently cultured on a Transwell insert on a decellularized matrixwith media collection every 48 hours. The culture was performed for 28days in the presence of varying concentrations of steroid hormones. H&Estaining and DAPI staining of the 3D myometrial units revealed thetissue architecture of the matrix as well as the presence of myometrialcells within the matrix.

Experiments were conducted during development of embodiments of thepresent invention to test the response of the endometrium on thedecellularized matrix to drug stimulus. Testosterone levels were addedduring the last 14 days of the 28 day cycle and cell behavior wasmonitored (FIG. 20 (right)). Cell survival experiments using a Wst-1assay in endometrial cells cultured in 2D over 72 hours revealed notoxicity of testosterone on the cells (FIG. 20 (left)). Experiments arealso conducted to grow myometrium on decellularized matrix (FIG. 54), totest universal media of the culture of multiple uterine cell types, totest response of uterine cell culture system to drug exposure, tomonitor the levels of secreted factors over the 28-day cycle, toidentify cellular markers and their levels over the 28-days cycle, etc.

Example 5 Ovarian Cultures in 3D Microphysiologic Setting

Experiments were conducted during development of embodiments of thepresent invention to encapsulate primary follicles in a microfluidicculture system, such that the 3D structure of the follicles ismaintained, and the follicles mimic in vivo ovarian responses, and thefollicles produce ovarian steroid hormones for downstream cultures inthe system.

Alginate hydrogel was used to encapsulate multiple primary follicles inthe microfluidic culture system, which maintains the 3D structure offollicle and recapitulates all key events of the folliculogenesis andoogenesis, including the follicle development from pre-antral to antralstage and oocyte meiotic and developmental competence acquisition(Hornick, et al., 2013). After the treatment with hCG, the maturefollicle undergoes ovulation and the oocyte initiates the second meiosisand extrudes the first polar body. These MII eggs are developmentalcompetent and can be fertilized through in vitro fertilization. Thefertilized eggs may develop to blastocyst stage in vitro, and give livebirth if they are transferred to a recipient host (FIG. 22).

Follicles are typically cultured in 96 or 24 wells cell culture plate,and exogenously added follicle stimulating hormone (FSH) is used tostimulate the follicle growth and human chorionic gonadotropin (hCG) isused to trigger ovulation. Systems described herein connect thedifferent female reproductive tissues together, thereby follicles arecultured in a connected (e.g., microfluidic) system which the ovarianculture is in fluid communication with the other reproductive tissues.In this way, the whole female reproductive tract connected in vitro, andthere is a real time delivery of follicle secreted hormones todownstream tissues. To achieve such a system, the traditional staticculture is recapitulated in the dynamic multi-culture system (FIG. 23).

In some embodiments, a ovarian culture subsystem comprises one or moreculture volumes (e.g., microphysiologic system (MPS) modules),overlaying a fluidic interface (e.g., allowing fluid communicationbetween culture volumes (e.g., with other ovarian culture volumes and/orwith other tissue subsystems), which in turn overlays an actuatorinterface. Such a design, or other suitable designed described hereinallow, for example: materials (e.g., media, cells, reagents, etc.) to besterilely introduced into the culture volumes (e.g., directly, via thefluidic interface, etc.), unidirectional and/or recirculated flow, floworiented toward the follicular subsystem, sufficient mixing of media,adjustable flow rate (e.g., in and/or out), collection of passed media,support of culture in polymer matrix (e.g., alginate beads),handling/loading of materials, etc.

Experiments were conducted during development of embodiments of thepresent invention demonstrating that the microfluidic ovarian subsystemsupports the follicle growth in vitro. Secondary follicles were culturedfor up to 60-90 hours, with significantly increased estradiol production(FIG. 24) and the follicles retrieved from microfluidic system showedgood morphology (FIG. 25). After hCG treatment, significantly increasedprogesterone secretion was observed (FIG. 26), indicating the follicleundergoes the luteal phase. These results demonstrate that themicrofluidic culture system supports the in vitro follicle growth in thesame manner as static culture.

Experiments were conducted during development of embodiments of thepresent invention to demonstrate the stability of FSH and hCG in themicrofluidic system, which are very critical for the follicledevelopment. A 14 days pretest was done with growth medium but withoutfollicles in the microfluidic system. Results indicate that after 48hours incubation, both FSH and hCG are stable in the microfluidic systemand there is no degradation (FIG. 27). Therefore, the materials used forthe microfluidic ovarian culture module are good for the follicleculture and added hormones are stable in the microfluidic system.

During the human 28-day estrous cycle, the pituitary hormones regulatethe follicle development, and the ovarian hormones regulate thedownstream tissue, such as uterine endometrium. The follicle developmentincludes two phases, the follicular phase and the luteal phase (FIG.28). During the follicular phase, follicles develop from pre-antralstage to antral stage. After the LH surge, the follicle ovulates MII eggand develop into the corpus luteam which is called luteal phase. Thefollicle secretes estrogen during follicular phase and secretesprogesterone during luteal phase to maintain the physiological functionsof ovary and other female reproductive tissues. The microfluidic systemdescribed herein utilizes a 28 day in vitro follicle culture to mimicthe 28 days human estrous cycle in vitro in the microfluidic system.

Experiments were conducted during development of embodiments of thepresent invention in which primary follicles were isolated from 12 daysold mice and encapsulated in 5% alginate, multiple follicleencapsulation is used because the primary follicles need each other tosupport the follicle growth. Follicles were cultured for 14 days tomimic the follicular phase with the growth medium with FSH. On day 14,follicles were treated with hCG to trigger the ovulation and initiatethe luteal phase with the growth medium without FSH. On day 28, allfollicle are collected for histology and gene expression study. Folliclegrowth was performed in three groups: (1) 10 follicles in one alginatebead, cultured in a well of a 96-well plate in 100 μl growth medium, (2)20 follicles in two alginate beads cultured a well of a 24-wells platewith 700 μl growth medium to mimic the condition for microfluidicculture system, (3) microfluidic culture system, 20 primary follicles in2 alginate beads in each culture module (FIG. 29). For the staticculture, growth medium is collected and replaced every 48 hours. For themicrofluidic culture, the system pumps 40 μl per hour to the follicleculture modele, thereby adding 960 μl of fresh media each day. In thecollection pore for fresh passed media, the capacity is only 100 μl, itrepresents the recent media pumped from the follicle culture well (FIG.31). 70 μl is collected each day. In the waste reservoir, the mediarepresents the media passed during the past 24 hours (FIG. 30). Folliclegrowth in the static culture in both 100 and 700 μl culture groupsindicates that the multiple follicle encapsulation supports the folliclegrowth (FIG. 31). The follicle diameter is around 310 in 100 μl staticculture, and 330 in 700 μl static culture, and there is no significantdifference for the follicle growth between these two static culturestrategies. The survival rates are 83.33% and 81.25%, and there is nosignificant difference. Microfluidic system also supported folliclegrowth in both the follicular phase (day 1-14) and luteal phase (day14-28) (FIG. 32). Since the daily follicle monitoring is not availablein the microfluidic culture system used in these experiments, folliclescultured in different modules were collected and imagined on differentdays (day 0, day 6, day 14 and day 28) (FIG. 32). Results indicate thatmicrofluidic system also supported the follicle growth in both thefollicular phase (day 1-14) and luteal phase (day 14-28) in microfluidicculture system (FIG. 32). The granulosa cells luteinized into granulosalutein cells after the LH surge in vivo. If there is no fertilization,the corpus luteum (CL) degenerates into the corpus albicans, which is afibrous scar tissue. The histology data indicate that all folliclesretrieved from both static and microfluidic systems on day 28 showfibrous scar like tissue, which consistent with the morphologicalchanges as in (FIG. 33).

Since the ovarian steroid hormones are critical for the downstreamtissue function in female reproductive system, ovarian hormonessecretion in the microfluidic system was tested to determine whetherthis system could mimic the 28 day female menstrual cycle in vitro. Thehormone secretion data demonstrate the follicles cultured inmicrofluidic phenocopy the 28 day human menstrual cycle. During thefirst 14 days of follicular phase, follicles keep secreting estradiolwhich peaks on day 14 (FIG. 34 (top)). After the hCG treatment on day14, follicles initiate the luteal phase and secret more progesteronewhich peaks 2 days after the hCG treatment (FIG. 34 (bottom)).

Follicles cultured in microfluidic system secreted more estradiol andprogesterone compared to 100 μl static culture (FIG. 35). In staticculture, follicles in 700 μl culture system, which is used to mimic themicrofluidic culture module, grow significantly faster than that in 100μl culture system. Thus indicating that promoted follicle growth in themicrofluidic culture system contributes to the increased estradiol andprogesterone secretion (FIG. 36).

Experiments were conducted during development of embodiments of thepresent invention to compare culture media outlet designs (e.g., snorkelvs. W Spwy). Follicle hormone secretion levels were compared betweenthese two different designs (FIG. 37). Results indicate that there is nosignificant difference for the hormone secretion levels between Snorkeland W Spill Way outlet designs for all three hormones analyzed. Thesedata indicate that there is no efficiency difference for these twodesigns.

Experiments were conducted during development of embodiments of thepresent invention to compare the use of a waste reservoir and collectionpore (FIG. 38). Media from the collection pore has higher estradiolexpression level from day 10, suggesting more estradiol secretion uponthe follicle growth, especially upon the follicle antrum formation.Although the absolute progesterone expression levels are similar, thepeak for the reservoir culture shifted 2 days later compared to themedia from collection pore, suggesting the progesterone secretion peaksaround 48-72 h after the hCG treatment and then gradually decreased.

Experiments were conducted during development of embodiments of thepresent invention to compare hormone levels in an exemplary ovarianmicrofluidic subsystem to physiologic levels (FIG. 39). Results indicatethat estradiol and progesterone levels in microfluidic system werehigher than human physiological levels (7 folds in 24 h accumulatedmedia), and that testosterone secretion levels were similar to the humanphysiological level (0.5-2 nM).

Experiments conducted during development of embodiments of the presentinvention demonstrated that 28-day whole ovary culture supports folliclegrowth in vitro and produce MII eggs after the hCG treatment on day 14.Moreover, the in vitro cultured ovary shows similar hormone expressionpatterns as the follicle culture in both static and microfluidic system(FIG. 40).

Example 6 Fallopian Cultures in 3D Microphysiologic Setting

Experiments were conducted during development of embodiments of thepresent invention to culture human fallopian tissues in a microfluidicculture system, such that the cultured tissue responds to the menstrualcycle (See e.g., Eddie S L, Quartuccio S M, Zhu J, Shepherd J A, KothariR, Kim J J, Woodruff T K, Burdette J E. Three-dimensional modeling ofthe human fallopian tube fimbriae. Gynecol Oncol. 2014 Dec. 16; hereinincorporated by reference in its entirety).

Alginate matrix was utilized to support human fallopian fimbriae exvivo. Fimbriae were cultured with factors hypothesized to contribute tocarcinogenesis, namely; H₂O₂ (1 mM) a mimetic of oxidative stress,insulin (5 μg/ml) to stimulate glycolysis, and estradiol (E₂, 10 nM)which peaks before ovulation. Cultures were evaluated for changes inproliferation and p53 expression, criteria utilized to identifypotential precursor lesions. Further, secretory factors were assessedafter treatment with E₂ to identify if steroid signaling induces apro-tumorigenic microenvironment.

3D fimbriae cultures maintained normal tissue architecture up to 7 days,retaining both epithelial subtypes. Treatment of cultures with H2O2 orinsulin significantly induced proliferation. However, p53 stabilizationwas unaffected by any particular treatment, although was induced by exvivo culturing. Moreover, E2-alone treatment significantly induced itscanonical target PR and expression of IL8, a factor linked to pooroutcome.

Tissue Collection

Fallopian fimbriae were collected with consent prior to surgery at theUniversity of Illinois at Chicago (UIC IRB #2012-0539). Patientsutilized in this study were undergoing salpingectomy for a variety ofgynecological purposes. Resulting tissues were deemed morphologicallynormal and considered benign as determined by gross examination by theUniversity of Illinois at Chicago Pathology Department. A total of 12samples from patients ranging from 28-62 years of age (average age of43).

3D Culture Optimization and Treatment

Tissues were micro-dissected in alpha-MEM (Gibco, Carlsbad, Calif.) with1% penicillin-streptomycin (Invitrogen, Carlsbad, Calif.). Individualfimbriae were separated into ˜50 mm³ pieces. A portion of the tissue wasfixed in 2% paraformaldehyde for use as an uncultured control. Foroptimization studies, fimbriae were cultured without matrix,encapsulated in 0.5% alginate, or encapsulated in 0.5% alginate with 1mg/ml collagen and 0.1% fibronectin, as previously described for murinecultures (King et al. J Vis Exp. 2011; herein incorporated by referencein its entirety). For subsequent treatments, 0.5% alginate encapsulatedfimbriae were randomly assigned to treatment groups, with at least fivefimbriae per condition, per patient, in a 24-well plate containingalpha-MEM and 1% penicillin-streptomycin. Tissues were treated with 1μl/ml ethanol (vehicle), 10 nM E₂ (Sigma-Aldrich, St. Louis, USA), 1 mMH₂O₂ (Fisher Scientific, Pittsburgh, Pa.), or 5 μg/ml insulin (via ITS(insulin; transferrin 5 μg/ml; selenite 5 ng/ml) Roche, Indianapolis,Ind.), and cultured for 2 or 7 days. Prior to fixation, fimbriae werelabeled with 10 μM bromodeoxyurine (BrdU, Sigma-Aldrich) for 24 hours todenote proliferating cells. Fimbriae cultures were fixed (2%paraformaldehyde) followed by dehydration in ethanol and xylene, andembedment in paraffin.

Tissue Preparation and Immunohistochemistry

Sections (5 μm) were cut and stained via hematoxylin and eosin formorphological analysis or immunohistochemistry was performed to localizeproteins of interest as previously described (King et al. Endocr RelatCancer. 2011. 18:627-42; herein incorporated by reference in itsentirety). Slides were rehydrated through an ethanol gradient, prior to0.1M sodium citrate retrieval and peroxidase block. Tissues probed forBrdU were exposed to 4M HCl and 0.1M NaB₄O₇ (Fisher Scientific) todenature DNA. All immunohistochemical reagents were obtained from VectorLaboratories, Inc (Burlingame, Calif.) unless otherwise stated. Tissueswere blocked in 3% bovine serum albumin (Gemini, West Sacramento,Calif.)-TBS/10% serum and incubated with a primary antibody 1:50acetylated tubulin (Cell Signaling, Cambridge, Mass.); 1:200 BrdU(AbCam, Cambridge, Mass.); 1:100 cytokeratin 8 (CK8, DevelopmentalStudies Hybridoma Bank, Iowa City, Iowa); 1:50 p53 (Santa Cruz, SantaCruz, Calif.); 1:100 PAX8 (Proteintech, Chicago, Ill.); 1:100 pH2AX(Cell Signaling); or 1:75 PR (Santa Cruz)) overnight at 4°. Tissues werewashed in TBS-0.1% Tween and incubated with a secondary antibody(1:200), before being probed with ABC peroxidase standard, followed bydetection with 3,3′-diaminobenzidine (DAB) and counterstained withhematoxylin.

Image Capture and Analysis

Immunohistochemistry images were taken via a Nikon E600 microscope,DXM1200 digital camera and NIS Elements software (Nikon Instruments,Melville, N.Y.). For proliferation analysis, concurrent sections werestained for CK8 and BrdU. BrdU sections were imaged and epithelial cells(CK8 positive) were quantified for proliferation via ImageJ software(NIH, Bethesda, Md.). At least three fimbriae with 200 or more FTE werequantified for each treatment. Analysis of p53 staining was similar,with at least three fimbriae per treatment, per patient analyzed.Samples with p53 expression were quantified utilizing adjacent sectionsstained for the secretory cell marker PAX8 in a qualitative manner asdescribed.

ELISA

IL8, VEGF-A, and FGF2 were detected in fallopian culture medium byenzyme-linked immunosorbent assay for human IL8 (EMD Millipore,Billerica, Mass., USA), VEGF-A (RayBiotech, GA, USA), or FGF2 (Abcam)respectively using the manufacturers' protocols. The sensitivity forIL8, VEGF-A, and FGF2 are 4.4 pg/ml, 10 pg/ml, and 2 pg/ml,respectively. Results were normalized to total protein content asdetermined by western blotting and Ponceau staining to account fordifference in tissue size between treatment groups. Conditioned medium(20 μl) was run on a 10% SDS-PAGE gel and transferred to nitrocellulosemembrane (Fisher Scientific). Ponceau (Sigma-Aldrich) staining andsubsequent densitometry via ImageJ software was performed.

3D Fimbriae Cultures Retain Tissue Architecture and Epithelial Subtypes

Optimization was performed to identify culture conditions that bestsupported fallopian architecture ex vivo. Previous studies identifiedalginate as an ideal matrix for the maintenance of baboon and murineovaries and oviducts (King et al. J Vis Exp. 2011; Xu et al. BiolReprod. 2011. 84:689-97; herein incorporated by reference in theirentireties), therefore 0.5% alginate was utilized to encapsulate humanfallopian fimbriae alongside fimbriae with no culture matrix, and 0.5%alginate supplemented with extracellular matrix (1 mg/ml collagen and0.1% fibronectin). Culture of human fimbriae revealed the alginatematrix maintained tissue architecture and cell morphology up to 7 days.Samples without matrix were also intact after 7 days, but had flatteningof the FTE in some areas. No additional benefit was seen withsupplementary ECM. Therefore, alginate hydrogel (0.5%) was utilized forsubsequent experiments.

Characterization after 2 and 7 days in culture indicated that alginatemaintained FTE viability and contact with the fallopian stroma (FIG.41). Both epithelial and stromal compartments appeared morphologicallysimilar to uncultured tissues, as indicated by H&E staining and theepithelial marker CK8. Further, unlike extended culture of fallopianepithelium in 2D where ciliated epithelia are lost, 3D fimbriae culturesretain both FTE subtypes; ciliated (acetylated tubulin) and secretory(PAX8), allowing for investigation of the role of both epithelialsubtypes in fallopian function and pathophysiology.

Insulin and H₂O₂ Induce Epithelial Proliferation in Fimbriae Cultures

Proliferation is an important aspect of normal physiology anduncontrolled proliferation is a hallmark of tumorigenesis. To clarifythe regulation of human FTE proliferation, ovulatory factorshypothesized to be involved in serous carcinogenesis were investigated(n=6). Insulin treatment, contained within the common culture supplementITS, induced FTE proliferation after both 2 and 7 days in culture(7.8±2.2% and 4.1±0.9% respectively) compared to vehicle control treated3D cultures (2.1±0.4% and 1.5±0.5%, FIGS. 42A and 42B). Proliferation incontrol cultures was similar to basal proliferation levels in normal invivo fimbriae (˜1-3%) (George et al. Clin Cancer Res. 2012. 18:6199-207;herein incorporated by reference in its entirety). The oxidative stressmimetic, H₂O₂, induced proliferation at 2 and 7 days (4.0±1.1% and2.2±0.5%, respectively). However, treatment with the steroid hormone E₂did not significantly affect epithelial proliferation (5.2±2.3% and2.4±0.5%, day 2 and day 7 respectively). FTE proliferation wasdetermined as a percentage of total FTE cells 24 hrs post-BrdU labeling(FIG. 42C). BrdU labeling results were supported by Ki67 immunostaining.

E₂ Regulates Fallopian Tissue and Induces Secretion of thePro-Tumorigenic Factor IL8

Although E₂ treatment did not induce proliferation, it was functional infimbriae samples, with the induction of its conical target, progesteronereceptor (PR) at both 1 nM and 10 nM concentrations compared to vehiclecontrol treated tissues (FIG. 43A). PR expression was weak and limitedto epithelial cells in control tissues and heightened in both the FTEand underlying fallopian stroma post-E₂ treatment, further demonstratingthe necessity of studying these cell types in association, assteroidogenic responses are induced in both tissue compartments. Toclarify the link between E₂ signaling and ovarian cancer risk, fallopiantissues were treated with 10 nM E₂ for 7 days and the conditioned mediumcompared to culture medium from samples treated with vehicle. Theseexperiments identified a significant increase in the pro-inflammatory,angiogenic cytokine interleukin 8 (IL8, FIG. 43B), in E₂ treated samples(22%±13% increase) compared to vehicle control (n=7). The modestincrease in IL8 induced by E₂ was specific, as other pro-tumorigenicfactors, including vascular endothelial growth factor (VEGF-A, 140%±75%increase compared to vehicle control, n=7, FIG. 43C), or fibroblastgrowth factor 2 (FGF2, 9%±8% decrease compared to vehicle control, n=6,FIG. 43D), which were not significantly altered post-treatment.Immunostaining was performed to determine localization of IL8 intissues. This demonstrated a weak diffuse staining pattern in 3Dcultured FTE samples that was primarily localized to the stromalcompartment of the cultures. These data confirm human fimbriae remainmetabolically active ex vivo.

p53 Stabilization is not Enhanced by Ovulatory Factors Ex Vivo

p53 induction in 3D cultures was evaluated, as its stabilization insecretory FTE is hypothesized to be a potential precursor to HGSC. Aswith proliferation studies, 3D fimbriae samples were treated for 2 and 7days with insulin, H₂O₂, and E₂ and compared with uncultured and vehiclecontrol treated tissues (n=8, FIG. 44A). Although no factor notablyinduced p53 stabilization at either time point, the ex vivo cultureprocess alone appeared to induce p53 expression. Similar to in vivo ‘p53signatures’, p53 stabilization was often limited to the secretory FTE,as noted by PAX8 expression (FIG. 44B). p53 expression was quantified bythe number of consecutive positive cells (as with the SEE-FIM protocol),with 12 cells denoting a full p53 signature. These data demonstrate anex vivo ‘forgery’ of the p53 signature can be produced that is similarto in vivo signatures originally defined in high-risk patients.Moreover, it was identified that these areas of p53 stabilization werenot always concomitant with DNA damage (pH2AX staining), indicating p53expression in the fallopian tube was not always in response to damagerepair. p53 and pH2AX staining were not apparent in uncultured normalfallopian tissues.

Example 7 Endocervical Cultures in 3D Microphysiologic Setting

Experiments were conducted during development of embodiments of thepresent invention to culture human endocervical tissues in amicrofluidic culture system, such that the cultured tissue is aphysiologic mimic of in vivo biology and responds to estrogen andprogesterone.

The endocervix has both anatomical and biological functions thatparticipate in the delicate balance between tolerance necessary forconception and protection from pathogens. Experiments were conductedduring development of embodiments of the present invention to develop arobust three-dimensional (3D) endocervix model that is a reliablerepresentation of the in vivo tissues and to identify the physiologicalresponses to changing levels of steroid hormones during a 28-day timeperiod. Such a model provides a endocervical subsystem for incorporationinto a microphysiologic system mimicking the entire female reproductivetract.

Human endocervical cells were grown on polystyrene scaffolds and themorphologic and hormonal responses of cultured cells were assessed inresponse to fluctuating levels of estradiol (E2) or progesterone (P4).Morphologically, the 3D cultures were composed of a mixed population ofcells including epithelial and stromal cells. Treatment with E2 and P4(day-28) increased cell growth and proliferation as compared to notreatment control. Cells expressed estrogen receptor (ER) andprogesterone receptor (PR) and produced both neutral and acidic mucins,including MUC16. In addition, a 45-plex Luminex assay identifiednumerous factors secreted and regulated by hormones. Specifically,Interleukin-1 beta (IL-1β) and Leukemia Inhibitory Factor (LIF)significantly decreased in the presence of E2 and P4 as compared to theno hormone control at day-26. Co-treatment with RU486 attenuated theprogesterone-driven inhibition of IL-1β and LIF secretion. A robust 3Dendocervical culture was developed and physiologic responses to themenstrual cycle mimic of estradiol and progesterone levels for a periodof 28 days were identified.

Tissue Collection

Endocervical tissue samples were collected from women undergoing routinehysterectomies at Northwestern University Prentice Women's Hospital(Chicago, Ill.), according to an IRB-approved protocol. Written consentwas obtained from all women included in the study.

Isolation and Expansion of Primary Human Endocervical Cells

The mucosal epithelium and the underlying stroma of the endocervix wereseparated from the muscular tissue, minced under sterile conditions into1- to 2-mm fragments and subjected to enzymatic digestion with 1.51mg/ml collagenase I (Invitrogen, NY) and 5 mg/ml DNase I (Sigma, MO) in20 ml Hanks' balanced salt solution (HBSS, Invitrogen, NY). Afterdigestion for 1-2 h at 37° C. shaker (100 rpm), cells were washed,resuspended in keratinocyte-serum free medium (KGM-Gold, KeratinocyteGrowth Medium Bullet Kit, Lonza, N.J.), plated on plastic culture dishesand cultured at 37° C. in a humidified atmosphere containing 5% CO2.Media was changed every 2-3 days.

3D Cell Cultures and Hormonal Treatment

A commercially available, highly porous polystyrene scaffold, Alvatex®(Reinnervate Ltd, Sedgefield, UK) was used for 3D cultures. Themembranes are 200 microns thick with pore sizes of 36-40 microns and the12-well TRANSWELL format was used. Scaffolds were incubated with sterilefiltered EtOH (70%) for 5 min as a pretreatment, washed twice withmedium and left in medium. Primary endocervical cells were trypsinizedfrom 2D cell cultures and each scaffold TRANSWELL was seeded with 2×106cells in a total volume of 100 μl medium (FIG. 1). The cells wereallowed to attach for 1 h and media was gently added to the lowerchamber for complete coverage with 3.5 ml media. Media was changed every48 h.

To represent a 28-day human menstrual cycle in vitro, a stepwise hormoneregimen was added to the cells. For the first 7 days, cells wereincubated with 0.1 nM E2, followed by 1 nM E2 for the next 7 days (FIG.45). Then cells were treated for 1 nM E2 and 10 nM P4 for an additional7 days, followed by 0.1 nM E2 and 50 nM P4 for 5 days. Then mediawithout hormones were added for the final 2 days for a total cultureperiod of 28 days. Media was collected every 48 h and stored at −20° C.For PR antagonist studies, cells were treated with 100 nM RU486 (Sigma,MO) which was added on day-21. To visualize the cells on scaffold, cellswere infected with the adenovirus containing pAD-eGFP-RLC (Cell ImagingCore, Northwestern University) (Khuon et al. Journal of cell science123:431-440; herein incorporated by reference in its entirety) by adding2 μl of amplified virus to 3.5 ml of media per TRANSWELL for 24 h.

Cell Viability

The Alamar Blue Cell Viability Assay (Invitrogen, NY) that measuresmetabolic activity was used to measure cell viability. 350 μl of AlamarBlue reagent was added directly to the cells at the end of 28-dayhormonal treatment. Cells were incubated at 37° C., 5% CO2 for 4 h inthe dark and the fluorescence was measured at 570/585 nm.

Immunohistochemistry

Scaffolds were washed twice in PBS, covered with histogel (FisherScientific, MA) and fixed in 4% paraformaldehyde for 5 h. Processing andhematoxylin and eosin (H&E) staining was performed at the NorthwesternUniversity Center for Reproductive Sciences Histology Core (Chicago,Ill.). Fixed cells were processed using an automated tissue processor(Leica) and embedded in paraffin. Serial sections were cut 10 μm thick,mounted on glass slides and stained with H&E using a Leica AutostainerXL (Leica Microsystems). Antigen retrieval was performed by treatmentfor 10 min in buffer containing 0.05% Trypsin and 1% CaCl2 at pH 7.6.After blocking, sections were incubated with primary antibodies to PR(1:200, Dako, Calif.), ER (1:100, Abgent, Calif.), vimentin (1:200,Abcam, MA), pan cytokeratin (1:500, Abcam, MA), Ki67 (1:100, Abcam, MA)and MUC16 (1:100, Abcam, MA) overnight at 4° C. Slides were rinsed andHRP-conjugated secondary antibodies were applied for 30 min. HRPactivity was detected using 3,3′-Diaminobenzidine (DAB) in 1:50 dilution(Dako, Calif.) as substrate for 30-60 sec and then counterstained withhematoxylin. Sections incubated with dilution buffer without primaryantibody were used as the negative control. For immunofluorescentstaining, antigen retrieval was performed by treatment for 10 min inbuffer containing 0.05% Trypsin and 1% CaCl at pH 7.6. Sections werewashed 2× in TBST, blocked with protein block for 30 min (Dako, Calif.)and incubated with primary antibodies described above. Slides wererinsed in TBST and incubated with fluorescent secondary antibodies(1:250; Invitrogen, NY) for 1 h. Slides were visualized and imagescaptured with Leica DM5000B Microscope.

Staining for Neutral and Acidic Mucins

Sections stained with Periodic Acid Schiff (PAS) (Abcam, MA) werepreviously deparaffinized and oxidized in 1% periodic acid for 10 min,followed by several rinses in distilled water. Staining was carried outin Schiff's reagent at 4° C. for 30 min followed by rinsing in distilledwater. Sections were then counterstained with hematoxylin for 2 min,washed, dehydrated and mounted. In addition, deparaffinized and hydratedsections were treated with acetic acid solution for 3 min, stained withAlcian blue (Abcam, MA) for 30 min, followed by a wash in running tapwater for 10 min. After counterstaining with Safranin O solution for 5min, sections were washed, dehydrated, and mounted.

Cytokine/Chemokine Quantitation

ProcartaPlex Human Cytokine/Chemokine/Growth Factor Panel 1 (45-plex)(Affymetrix eBioscience, CA) was used to measure levels of secretedcytokines and chemokines. For quantification, duplicate standardsproduced a curve for each analyte from which concentrations of sampleswere extrapolated. Medium alone was used to establish background levelsof cytokines and chemokines. The levels of LIF, IL-1β (Abcam) and MUC16(R&D) were validated using single ELISA assays and performed accordingto manufacturer's protocol.

Establishment of an Ex Vivo 3D Endocervix

Human endocervix tissues obtained post-surgery were enzymaticallydigested and expanded in 2D cultures to obtain a sufficient number ofcells (FIG. 46). Two million cells were seeded on the highly porouspolystyrene scaffolds, Alvetex® membranes (FIG. 46) (Knight et al.Methods in molecular biology 695:323-340; Bokhari et al. Journal ofanatomy 211:567-576; Stevanato & Sinden Stem cell research & therapy5:49; herein incorporated by reference in their entireties) andimmediately treated with E2 and P4 in a stepwise fashion to mimic a28-day menstrual cycle in vitro (FIG. 45A). At 14-day (E2 only) and28-day (E2+P4) time points, viable endocervical cells were present, asvisualized with GFP florescence (FIG. 45B). The cell viability assaydemonstrated viable cells at 28-days. This assay also showed a subtlebut significant increase in cell viability with E2+P4 treatment at day28 compared to vehicle treated control (p<0.05) (FIG. 45C). These datademonstrate that endocervical cells cultured on the 3D scaffolds surviveand grow in response to a 28-day culture with hormones.

Histological Analysis of 3D Cultures

H&E staining of the 3D cultures revealed the presence of a mixedpopulation of cells (FIG. 47). Pan cytokeratin and vimentin stainingshowed both epithelial and stromal cells, respectively, present in theendocervical cultures at day-14 (E2) and day-28 (E2+P4). In addition,cells expressed both PR and ER, which increased in the presence of E2and P4 as compared to control. Furthermore, the Ki67 staining wasevident in the cultures with increased staining in the presence of E2,or E2 and P4 as compared to control (FIG. 47) indicative of activeproliferation of cells, even after 28 days of culture.

Expression and Secretion of Endocervical Mucus

One of the major functional properties of the endocervix is theproduction and secretion of mucus. Immunohistochemical staining showedproduction of the mucin, MUC16 (FIG. 47C). Estrogen and progesterone didnot appear to affect levels of MUC16 compared to control cultures. Inaddition, levels of MUC16 secreted into the culture media did notsignificantly change at day-26 and day-28 of the hormone treatmentregimen compared to untreated cultures (FIG. 47D). Moreover, theprogesterone receptor antagonist, RU486 in the presence of E2 and P4 didnot significantly alter levels of secreted MUC16 (FIG. 47D), indicatingthat hormones were not modulating the production and secretion of MUC16in the 3D endocervix cultures. In contrast, the neutral mucins, asmeasured by Periodic Acid Schiff (PAS) staining, increased with E2 andP4 (day-28) treatment compared to control or E2 (day-14) treatments(FIG. 47C). This stain also detected the presence of goblet cells at theend of the 28-day hormonal treatment (FIG. 47C). The acidic mucins weredetected using the Alcian blue stain. In contrast to the PAS stain, nodifference in the levels of acidic mucins with the hormonal treatmentswas observed (FIG. 47C).

Cytokine/Chemokine Profile of 3D Cultures

The cytokine/chemokines secreted by the 3D endocervical cultures weremeasured using a 45-Plex Luminex assay. Levels of factors secreted intothe culture media at day 14, 26 and 28 were measured during the stephormone treatment. Out of 45 factors, 36 were detectable by this assay.There was a wide range in concentrations of factors secreted by the 3Dcultures; 17 factors measured less than 25 pg/ml consistently within thereplicates. Due to the variation among patient samples, data wasexpressed as fold changes of hormone treated with its respective control(vehicle treated) (FIG. 48A). Fold changes were then compared betweenthe three time points, day 14, 26 and 28. Seven factors, IL27, IP10,IL-1β, MCP1 HGF, VEGFA, and LIF showed fold changes that werestatistically different between day 14 and day 28 (IL27, IP10, VEGFA andLIF) or between day 26 and day 28 (IL-113, MCP1, HGF, and LIF).Validation of two factors, IL-1β and LIF using single ELISA assays anddifferent patient samples, demonstrated that E2 and P4 treatmentdecreased the secretion of these factors at day 26 (FIG. 48B). Treatmentof cultures with RU486 starting from day 21 attenuated the inhibition ofIL-1β and LIF that occurred with E2 and P4, demonstrating specificregulation by progesterone through its receptor (FIG. 48B).

Example 8 Ectocervical Cultures in 3D Microphysiologic Setting

Experiments were conducted during development of embodiments of thepresent invention to culture human ectocervical tissues in amicrofluidic culture system, such that the cultured tissue responds toovarian hormones.

The ectocervix epithelium comprises four primary layers: superficial,intermediate, parabasal, and basal layers (FIG. 49). The cells of theselayers undergo changes throughout the menstrual cycle (FIG. 50). A 3Dculture subsystem was designed comprising cells of each of the fourlayers. The culture subsystem recapitulates these changes byproliferation and differentiating in response to estrogen. Additionally,progesterone receptor expression increases in response to estrogen,whereas the secretion of antimicrobial peptides will decrease inresponse to estrogen.

Experiments were conducted to establish static 3D cultures of primaryepithelial cells from the ectocervix (FIG. 51):

a. Collagen plug with J2-3T3 fibroblasts

-   -   1. Detached J2-3T3 cells with 0.05% trypsin and count cells        −400,000 cells per 1.5 ml plug.    -   2. Spun down appropriate amount of cells and resuspend in 1/10        volume reconstitution buffer (1.95 ml).    -   3. Added 1/10 volume 10×DMEM (1.95 ml) and rocked gently to mix.    -   4. Added collagen to a final concentration of 4 mg/ml and rocked        gently.    -   5. Added 0.5 N NaOH until color resembled watermelon        pink—neutral.    -   6. Added ddH2O to reach final volume (19.5 ml).    -   7. Added 1.5 ml collagen solution to each well of a 12-well        plate.    -   8. Incubated plates at 37° C. for 30 minutes to allow collagen        to polymerize.    -   9. Added 3 ml J2-3T3 media to each well.    -   10. Incubated at 37 C for 2 days. Collagen plug was viable for        up to 4 days. Media on plug changed every other day.

b. Seeding epithelial cells on collagen plug

-   -   1. Removed existing J2-3T3 media from collagen plugs, taking        care not to touch the collagen.    -   2. Added 2 ml E media (below)+5 ng EGF to each well to submerge        collagen plug.    -   3. Removed feeder layer from 2D cell cultures, then trypsinized        and counted cells    -   4. Resuspended cells in 1 ml/well E media.    -   5. Added 1 ml of cell suspension to each well (for a total of 3        ml per plug).    -   6. Incubate at 37 C until confluent, changing media every day.

3D ectocervix raft culture: liquid-air interface

-   -   1. Removed media from collagen plug, taking care not to touch        the collagen.    -   2. Removed collagen plugs from wells by inserting sterile        spatula between the well and plug in an up and down motion on        all side of plug, and then carefully scooped underneath plug and        placed it on metal grid in a 60 mm plate.    -   3. Added fresh E media+EGF to each of the 60 mm plates up to the        bottom of the grid.    -   4. Changed media every 2 days until harvest.        E media (1 L): 458.5 ml DMEM HG, 458.5 ml DMEM:F-12, 10 ml E        cocktail mix (below), 200 μl gentamicin (10 μg/ml), 1 ml        Amphotericin B (0.25 μg/ml, 20 ml 4 mm L-Glut (200 mM), 1 ml        hydrocortisone (0.4 μg/ml).        E cocktail mix: 180 μM adenine, 5 μg/ml human recombinant        insulin, 5 μg/ml human apo-transferin, 5 μg/ml triiodothyronine,        T3 and 1 ml cholera toxin (10 ng/ml).        The basal and parabasal layers had formed by day 7, with the        more superficial layers beginning to form in ROCK inhibitor        treated cells. By 14 days, all 4 layers were apparent in both        the primary and ROCK inhibitor treated cells (FIG. 52).

Example 9 Decellularizing Human Ectocervix Tissue

Experiments were conducted during development of embodiments of thepresent invention to decellularize human ectocervix tissue according tothe following exemplary protocol:

-   -   1. Wash tissue with PBS+antibiotics to remove contaminants.    -   2. Cut a small piece and flash freeze in LN2 for later DNA        comparisons.    -   3. Cut a small piece and fix in 4% PFA for histology.    -   4. Place remaining tissue in 50 ml 0.1% SDS on a rocker at RT.    -   5. Place tissue in fresh 50 ml 0.1% SDS and place on rocker at        RT for an additional 1-2 days.    -   6. Place tissue in 50 ml PBS and place on rocker in cold room        overnight.    -   7. Replace with 50 ml fresh PBS and continue washing overnight        at 4 C.    -   8. Place tissue in PBS with 1 mg/ml BSA and 0.05% sodium azide        and store at 4 C until needed.

Example 10 Differentiation of iPSCs into Intermediate Mesoderm Lineage

Experiments were conducted during development of embodiments of thepresent invention to differentiate induced pluripotent stem cells(iPSCs) according to the following exemplary protocol:

Embryoid Body Formation:

-   -   1. iPSCs are 85-90% confluent before embryoid body formation,        and there is less than 10% differentiation per well. Embryoid        bodies are formed in the same size well that the confluent iPSCs        were originally plated (1:1).    -   2. Wash iPSCs were versene, then add 1 ml of versene to each        well and incubate at room temperature for 6 minutes.    -   3. Lift cells in E8 media and pool in a 15 ml conical.    -   4. Centrifuge at 1000 rpm for 5 minutes to form pellet.    -   5. Add 2 ml/well of E8 with PVA to non-adherent 35 mm plates.    -   6. Resuspend cell pellet in 0.5 ml/well E8 with PVA.    -   7. Add 1 ul/well ROCK inhibitor.    -   8. Add 0.5 ml cell suspension drop wise to prepared 35 mm        plates.    -   9. Incubate at 37° C. overnight—embryoid bodies will form within        two days.

Embryoid Bodies to Intermediate Mesoderm

-   -   1. Carefully remove media from embryoid bodies by tilting the        plate to a 45 degree angle and allowing embryoid bodies to        settle at the bottom. Aspirate as much media as possible without        disturbing embryoid bodies.    -   2. Replace with 2 ml maintenance media with BMP4 for 1 day.    -   3. Replace media with 2 ml intermediate mesoderm differentiation        media, changing media every day for 3 days.    -   4. Plate cells on fibronectin coated plate in intermediate        mesoderm media, changing media daily until morphologically        desirable.    -   5. Analyze for IM and pluripotent markers.

Example 11 Harvesting 3D Ectocervix Rafts

The following exemplary protocol was used for harvesting 3D ectocervixrafts produced, for example, as described above:

-   -   1. Discard media (or save media if needed for future analysis).    -   2. Flood raft with PBS (with calcium) to completely submerge the        entire raft.    -   3. Gently remove collagen plug from grid, without disturbing the        epithelial layer. Use forceps to gently pick the plug around the        edges, or use scissors to cut away the plug from the raft.    -   4. Once collagen plug is completely removed and floating, push        to the side and remove grid; do not flip or disturb the collagen        plug.    -   5. While holding plug to the side, remove PBS from dish.    -   6. Cut three parallel sections: one for RNA, OCT and protein.        The very center section will be for OCT.    -   7. Set aside the middle piece for OCT and remove the epithelial        layers from the two side pieces by using forceps to gently pull        the cell sheet away from the collagen.    -   8. Place epithelial sheets in either Trizol for RNA processing        or Urea buffer for protein analysis and let sit several minutes.    -   9. Trim the section for OCT, removing the ends and leaving a        piece that will fit nicely in the OCT tissue chamber.    -   10. Fill chamber about ½ full with OCT, place on metal block in        LN2 until slightly (but not completely) frozen—will be white but        still transparent at the top.    -   11. Place tissue onto OCT in chamber, being careful not to        stretch it. Make sure the tissue is not folded and is in the        correct orientation.    -   12. Place on metal block in LN2 and fill the chamber with OCT,        covering tissue, without air bubbles.    -   13. Leave on metal block until completely frozen, while        completing RNA and protein    -   14. Use needles and syringes to break down the tissue for RNA        and protein:    -   RNA: 18 gauge, then 22 gauge    -   Protein: 18 gauge, then 22 gauge, then 26 gauge    -   15. Store all at −80 C until needed.

Example 12 Primary Ectocervix Cell Culture

The following exemplary protocol was used for 3D culturing of 3Dectocervix cells:

Epithelial Cell Isolation

-   -   1. Wash tissue in PBS+antibiotics to remove any contaminants.    -   2. Cut tissue into small pieces (<5 mm) and place in 60 mm dish        with 3 ml dispase in 4 C fridge overnight.    -   3. Remove epithelial layer by using two pairs of forceps, one to        anchor the tissue, and one to gently peel off epithelial layer.    -   4. Place epithelial layers flat in 4 ml 0.25% trypsin in a 60 mm        plate for 10 mins at 37° C.    -   5. Add 500 μl FBS to neutralize trypsin.    -   6. Add 5 ml PBS and pipette up and down to release cells.    -   7. Transfer to 15 ml conical and centrifuge at 1000 rpm for 5        minutes.    -   8. Resuspend in FAD or keratinocyte media and plate on J2-3T3        feeder layer. Change media the day after plating Change feeder        layer and media every 2 days after plating. Follow differential        trypsinization protocol for passaging and changing feeder layer.

Feeder Layer Preparation:

-   -   1. Treat J2-3T3 cells with Mitomycin C for 2-2.5 hours.    -   2. Wash cells 3× with PBS to remove mitomycin C.    -   3. Add 1 ml 0.05% trypsin and incubate at RT for 2 minutes.    -   4. Neutralize with FAD media and transfer to a 15 ml conical.    -   5. Centrifuge at 1000 rpm for 5 minutes.    -   6. Resuspend in FAD media and distribute to plates for        epithelial cell co-culture.        Stromal Fibroblast Isolation:    -   1. After removing epithelial layer from tissue with dispase, cut        remaining tissue into smaller pieces (<2 mm) and place in 100 mm        plate for 10 minutes at RT to allow tissue to stick to plate.    -   2. Add 1 drop of 0.25% trypsin to each piece (about 1 ml total)        and incubate at 37° C. for 2.5-3 hours (Alternative: use 0.05%        trypsin and increase incubation time to 8 hours).    -   3. Flood plate with 7 ml J2-3T3 media.    -   4. Change media the following day and then every 2 days after        that.    -   5. Remove tissue once fibroblasts are growing out from the        tissue pieces onto the plate.    -   6. Passage with 0.25% trypsin once cells reach confluency.        Differential Trypsinization:    -   1. Remove media and replace with 4 ml Versene for 5-10 minutes        at RT.    -   2. Pipette versene to remove fibroblasts and confirm detachment        with microscope.    -   3. Remove versene and add 1 ml 0.05% trypsin for 5-7 minutes at        37 C. Confirm detachment under microscope.    -   4. Neutralize trypsin by adding 3-4 ml FAD media, and transfer        to 15 ml conical.    -   5. Centrifuge at 1000 rpm for 5 minutes at RT.    -   6. Resuspend cells in FAD media and plate on freshly prepared        feeder cells.        The foregoing embodiments and examples are intended only as        examples. No particular embodiment, example, or element of a        particular embodiment or example is to be construed as a        critical, required, or essential element or feature of any of        the claims. Various alterations, modifications, substitutions,        and other variations can be made to the disclosed embodiments        without departing from the scope of the present application,        which is defined by the appended claims. The specification,        including the figures and examples, is to be regarded in an        illustrative manner, rather than a restrictive one, and all such        modifications and substitutions are intended to be included        within the scope of the application. Steps recited in any of the        method or process claims may be executed in any feasible order        and are not limited to an order presented in any of the        embodiments, the examples, or the claims.

REFERENCES

The following references, as well as those included above, are hereinincorporated by reference in their entireties.

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We claim:
 1. A microphysiologic system comprising: (a) an ovarianfollicle 3D cell culture subsystem comprising at least a first cell typein 3D culture and a culture media for said first cell type; and (b) afallopian tube 3D cell culture subsystem comprising at least a secondcell type in 3D culture and a culture media for said second cell type;wherein the ovarian follicle 3D cell culture subsystem and the fallopiantube 3D cell culture subsystem are in unidirectional fluid communicationsuch that fluid from the ovarian follicle 3D cell culture subsystemflows downstream to the fallopian tube 3D cell culture subsystem.
 2. Themicrophysiologic system of claim 1, further comprising: (c) a uterine 3Dcell culture subsystem comprising at least a third cell type in 3Dculture and a culture media for said third cell type; wherein thefallopian tube 3D cell culture subsystem and the uterine 3D cell culturesubsystem are in unidirectional fluid communication such that fluid fromthe fallopian tube 3D cell culture subsystem flows downstream to theuterine 3D cell culture subsystem.
 3. The microphysiologic system ofclaim 2, further comprising: (d) an endocervical 3D cell culturesubsystem comprising at least a fourth cell type in 3D culture and aculture media for said fourth cell type; wherein the uterine 3D cellculture subsystem and the endocervical 3D cell culture subsystem are inunidirectional fluid communication such that fluid from the uterine 3Dcell culture subsystem flows downstream to the endocervical 3D cellculture subsystem.
 4. The microphysiologic system of claim 3, furthercomprising: (e) an ectocervical 3D cell culture subsystem comprising atleast a fifth cell type in 3D culture and a culture media for said fifthcell type; wherein the endocervical 3D cell culture subsystem and theectocervical 3D cell culture subsystem are in unidirectional fluidcommunication such that fluid from the endocervical 3D cell culturesubsystem flows downstream to the ectocervical 3D cell culturesubsystem.
 5. The microphysiologic system of claim 4, wherein factorssecreted from cells in the ovarian follicle 3D cell culture subsystemflow downstream to the fallopian tube, uterine, endocervical, and/orectocervical 3D cell culture subsystems, factors secreted from cells inthe fallopian tube 3D cell culture subsystem flow downstream to theuterine, endocervical, and/or ectocervical 3D cell culture subsystems,factors secreted from cells in the uterine 3D cell culture subsystemflow downstream to the endocervical and/or ectocervical 3D cell culturesubsystems 3D cell culture subsystem, and factors secreted from cells inthe endocervical 3D cell culture subsystem flow downstream to theectocervical 3D cell culture subsystem.
 6. The microphysiologic systemof claim 1, wherein the ovarian follicle 3D cell culture subsystemcomprises one or more of granulosa cells, theca cells, and oocytes. 7.The microphysiologic system of claim 6, wherein the ovarian follicle 3Dcell culture subsystem comprises one or more ovarian follicles in 3Dculture.
 8. The microphysiologic system of claim 7, wherein the ovarianfollicles are polymer-encapsulated.
 9. The microphysiologic system ofclaim 8, wherein the polymer comprises alginate.
 10. Themicrophysiologic system of claim 7, wherein the ovarian follicles remainviable for at least one simulated menstrual cycle.
 11. Themicrophysiologic system of claim 7, wherein the ovarian folliclesrespond to FSH and hCG stimulation by producing estrogen andprogesterone in a pattern that mimics the human menstrual cycle.
 12. Themicrophysiologic system of claim 11, wherein said estrogen andprogesterone pass by unidirectional fluid communication to the fallopiantube 3D cell culture subsystem.
 13. The microphysiologic system of claim12, wherein cells of the fallopian tube 3D cell culture subsystemrespond to said estrogen and/or progesterone with one or more ofcilliary beating, increased oviductal glycoprotein 1 (OVGP1) expression,and cilliary beat frequency (CBF) and/or insulin-like growth factor 1(IGF1) secretion.
 14. The microphysiologic system of claim 1, whereinthe fallopian tube 3D cell culture subsystem comprises one or both ofsecretory epithelial cells and ciliated epithelial cells.
 15. Themicrophysiologic system of claim 14, wherein the fallopian tube 3D cellculture subsystem comprises fallopian epithelium tissue pieces grown onmedia-permeable cell supports.
 16. The system of claim 14, wherein thefallopian tube system remains viable and maintains both secretory andciliated epithelium cell phenotypes for at least one simulated menstrualcycle.
 17. The microphysiologic system of claim 1, wherein the uterine3D cell culture subsystem comprises one or more of endometrialepithelial cells, endometrial stromal cells, myometrial smooth musclecells, and myometrial fibroblast cells.
 18. The microphysiologic systemof claim 1, wherein the endocervical 3D cell culture subsystem comprisesone or both of endocervical epithelial cells and endocervical stromalcells.
 19. The microphysiologic system of claim 1, wherein theectocervical 3D cell culture subsystem comprises one or both ofectocervical epithelial cells, J2-3T3 fibroblasts, and ectocervicalstromal cells.
 20. The microphysiologic system of claim 1, comprising amicrofluidic component that provides said unidirectional fluidcommunication.
 21. A microphysiologic system comprising: (a) an ovarianfollicle 3D cell culture subsystem comprising granulosa cells, thecacells, and/or oocytes in 3D culture; (b) a fallopian tube 3D cellculture subsystem comprising secretory epithelial cells and/or ciliatedepithelial cells in 3D culture; (c) a uterine 3D cell culture subsystemcomprising endometrial epithelial cells, endometrial stromal cells,myometrial smooth muscle cells, and/or myometrial fibroblast cells in 3Dculture; (d) an endocervical 3D cell culture subsystem comprisingendocervical epithelial cells and/or endocervical stromal cells in 3Dculture; and (e) an ectocervical 3D cell culture subsystem comprisingectocervical epithelial cells, J2-3T3 fibroblasts, and/or ectocervicalstromal cells in 3D culture; wherein the ovarian follicle 3D cellculture subsystem and the fallopian tube 3D cell culture subsystem arein unidirectional fluid communication such that fluid and factors fromthe ovarian follicle 3D cell culture subsystem flow downstream to thefallopian tube 3D cell culture subsystem; wherein the fallopian tube 3Dcell culture subsystem and the uterine 3D cell culture subsystem are inunidirectional fluid communication such that fluid and factors from thefallopian tube 3D cell culture subsystem flow downstream to the uterine3D cell culture subsystem; wherein the uterine 3D cell culture subsystemand the endocervical 3D cell culture subsystem are in unidirectionalfluid communication such that fluid and factors from the uterine 3D cellculture subsystem flow downstream to the endocervical 3D cell culturesubsystem; wherein the endocervical 3D cell culture subsystem and theectocervical 3D cell culture subsystem are in unidirectional fluidcommunication such that fluid and factors from the endocervical 3D cellculture subsystem flow downstream to the fourth ectocervical 3D cellculture subsystem.